Vaccine

ABSTRACT

The present invention is in the field of pneumococcal capsular saccharide conjugate vaccines. Specifically, the present invention relates to immunogenic compositions and vaccines comprising detoxified pneumolysin adsorbed onto aluminium phosphate and an improved process for the adsorption of detoxified pneumolysin onto aluminium phosphate. It additionally relates to the use of the immunogenic compositions and vaccines in the treatment or prevention of Streptococcus pneumoniae infection.

FIELD OF THE INVENTION

The present invention relates to immunogenic compositions and vaccinescomprising detoxified pneumolysin adsorbed onto aluminium phosphate andan improved process for the adsorption of detoxified pneumolysin ontoaluminium phosphate. It additionally relates to the use of theimmunogenic compositions and vaccines comprising detoxified pneumolysinadsorbed onto aluminium phosphate in the treatment or prevention ofStreptococcus pneumoniae infection.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive bacteriumresponsible for considerable morbidity and mortality (particularly ininfants and the elderly), causing invasive diseases such as bacteraemiaand meningitis, pneumonia and other non-invasive diseases, such as acuteotitis media. About 800,000 children die annually due to pneumococcaldisease, especially in emerging countries (0-Brien et al. 2009 Lancet374:893-902). The increasing number of antibiotic-resistant strains(Linares et al. 2010 Cin. Microbiol. Infect. 16:402-410) and theseverity of pneumococcal diseases make vaccination the most effectiveintervention.

The major clinical syndromes caused by S. pneumoniae are widelyrecognized and discussed in standard medical textbooks (Fedson D S,Muscher D M. In: Plotkin S A, Orenstein W A, editors. Vaccines. 4thedition. Philadelphia WB Saunders Co, 2004a: 529-588). For instance,Invasive Pneumococcal Disease (IPD) is defined as any infection in whichS. pneumoniae is isolated from the blood or another normally sterilesite (Musher D M. Streptococcus pneumoniae. In Mandell G L, Bennett J E,Dolin R (eds). Principles and Practice of Infectious diseases (5th ed).New York, Churchill Livingstone, 2001, p 2128-2147). Chronic obstructivepulmonary disease (COPD) is recognised as encompassing severalconditions (airflow obstruction, chronic bronchitis, bronchiolitis orsmall airways disease and emphysema) that often coexist (Wilson et al.,Eur. Respir. J. 2001; 17: 995-1007). Patients suffer exacerbations ofCOPD that are usually associated with increased breathlessness, andoften have increased cough that may be productive of mucus or purulentsputum (Wilson, Eur Respir J 2001 17:995-1007). COPD is definedphysiologically by the presence of irreversible or partially reversibleairway obstruction in patients with chronic bronchitis and/or emphysema(Standards for the diagnosis and care of patients with chronicobstructive pulmonary disease. American Thoracic Society. Am J RespirCrit Care Med. 1995 November; 152(5 Pt 2):S77-121). Exacerbations ofCOPD are often caused by bacterial (e.g. pneumococcal) infection (SethiS, Murphy T F. Bacterial infection in chronic obstructive pulmonarydisease in 2000: a state-of-the-art review. Clin Microbiol Rev. 2001April; 14(2):336-63).

Streptococcus pneumoniae, also referred to as pneumococcus, isencapsulated with a chemically linked polysaccharide which confersserotype specificity. There are more than 90 known serotypes ofpneumococci, and the capsule is the principle virulence determinant forpneumococci, as the capsule not only protects the inner surface of thebacteria from complement, but is itself poorly immunogenic. Ananti-polysaccharide antibody level has been regarded as predictive ofthe protection against invasive pneumococcal disease (Jodar et al.Vaccine, (21) 2003, p. 3264-3272). After initial licensure of a 7-valentconjugate vaccine containing serotypes 4, 6B, 9V, 14, 18C, 19F, 23F(PCV7), two pneumococcal conjugate vaccines (PCVs) designed to broadencoverage have been licensed. The 10-valent pneumococcal Haemophilusinfluenzae protein D conjugate vaccine (PCV10) contains serotypes 1, 4,5, 6B, 7F, 9V, 14 and 23F conjugated to nontypeable H. influenzaeprotein D, plus serotype 18C conjugated to tetanus toxoid and serotype19F conjugated to diphtheria toxoid. The 13-valent pneumococcalconjugate vaccine (PCV13) contains the PCV7 (4, 6B, 9V, 14, 18C, 19F,23F) serotypes plus serotypes 1, 3, 5, 6A, 7F and 19A, conjugated tocross-reactive material CRM197. It is an object of the present inventionto develop improved Streptococcus pneumoniae vaccines.

Pneumolysin (ply) is a 53 kDa thiol-activated cytolysin found in allstrains of S. pneumoniae, which is released on autolysis and contributesto the pathogenesis of S. pneumoniae. It is highly conserved with only afew amino acid substitutions occurring between the ply proteins ofdifferent serotypes. Pneumolysin is a multifunctional toxin with adistinct cytolytic (hemolytic) and complement activation activities(Rubins et al., Am. Respi. Cit Care Med, 153:1339-1346 (1996)). Thetoxin is not secreted by pneumococci, but it is released upon lysis ofpneumococci under the influence of autolysin. Its effects include, forexample, the stimulation of the production of inflammatory cytokines byhuman monocytes, the inhibition of the beating of cilia on humanrespiratory epithelial, the decrease of bactericidal activity andmigration of neutrophils, and in the lysis of red blood cells, whichinvolves binding to cholesterol. Expression and cloning of wild-type ornative pneumolysin is described in Walker et al. (Infect Immun,55:1184-1189 (1987)), Mitchell et al. (Biochim Biophys Acta, 1007:67-72(1989) and Mitchell et al (NAR, 18:4010 (1990)).

The present invention provides detoxified pneumolysin adsorbed ontoaluminium phosphate having improved properties and an improved processfor the adsorption of detoxified pneumolysin onto aluminium phosphate.The present inventors have found that by admixing detoxified pneumolysinand aluminium phosphate at a specific pH range a high level ofcompleteness of adsorption may be obtained (greater than 85%), and anadsorbed detoxified pneumolysin having desirable properties, for examplein relation to particle size, can be produced. The particle size of theadsorbed detoxified pneumolysin and level of adsorption can affectimmunogenicity; therefore, a particle size <10 μm and a high level ofadsorption (greater than 85%) are desirable. Furthermore, the presentinventors have found that it is advantageous to pre-adsorb detoxifiedpneumolysin onto aluminium phosphate according to the method ofinvention prior to mixing with other antigens. For example, pre-adsorbeddetoxified pneumolysin may be mixed with pre-adsorbed PhtD which hasbeen adsorbed onto aluminium phosphate under different conditions.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 Evaluation of Completeness of Adsorption. Compares completenessof adsorption of detoxified pneumolysin (dPly) onto aluminium phosphateunder different pHs and ratios of dPly:Al³⁺ (from aluminium phosphate)(i) pH5.5-6.1 and ratio 1:1, (ii) pH5.5-6.1 and ratio 1:2, (iii) pH5.5to 6.1, ratio 1:3, and (iv) pH6.5 and ratio 1:3. Two different antigenlots were tested: E-DPLY-P14 and DPLYADA007.

FIG. 2 Antigenicity. Shows Elisa recovery for dPly adsorbed ontoaluminium phosphate at pH5.5 to 6.1, ratio of dPly:Al³⁺ (from aluminiumphosphate) of 1:3. The bars correspond to the two different antigen lotsprepared according to the method of Example 1: the bars on the leftcorrespond to dPly E-DPLY-P14 and the bars on the right correspond todPly DPLYADA007. The dPly was stored either for 2 weeks at 4° C. (T2w4°C.) or 1 week at 4° C. followed by 1 week at 37° C. (T1w4° C.+1w37° C.).Note: this figure has been corrected to show that dPly DPLYADA007 had arecovery of 110% by Elisa after 1 week at 4° C. followed by 1 week at37° C. (T1w4° C.+1w37° C.).

FIG. 3 Particle size. Compares the percentage of particles of dPlyadsorbed onto aluminium phosphate less than 10 μm for under different pHand ratios of dPly:Al³⁺ (from aluminium phosphate): (i) pH5.5-6.1 andratio 1:1 and (ii) pH5.5 to 6.1, ratio 1:3. The bars from left to rightcorrespond to T0 (time=zero), T7d4° C. (7 days at 4° C.), T7d37° C. (7days at 37° C.), T10d4+6d37° C. (10 days at 4° C. and 6 days at 37° C.)and T21d4° C. (21 days at 4° C.). Data for the two different antigenlots is shown: dPly E-DPLY-P14 on the right and dPly DPLYADA007 on theleft.

DESCRIPTION OF THE INVENTION

The present invention provides an immunogenic composition comprisingdetoxified pneumolysin having a high level of adsorption (greater than85%) onto aluminium phosphate. The present invention also provides animproved process for adsorption of detoxified pneumolysin onto aluminiumphosphate.

Accordingly, in the first aspect of the present invention, there isprovided an immunogenic composition or vaccine comprising detoxifiedpneumolysin adsorbed onto an aluminium phosphate, wherein more than 85%(suitably more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%)of the detoxified pneumolysin is adsorbed onto the aluminium phosphate.

In another aspect, the present invention provides a process foradsorption of detoxified pneumolysin onto an aluminium phosphatecomprising the step of (i) admixing detoxified pneumolysin and thealuminium phosphate at a pH less than 6.5 (e.g. less than 6.4, less than6.3, less than 6.2, less than 6.1), suitably less than pH 6.0, forexample pH 5.0 to 6.2, pH 5.0 to 6.1, pH 5.2 to 6.2, pH 5.2 to 6.1, pH5.4 to 6.2, pH 5.4 to 6.1, pH 5.5 to 6.1, pH 5.4 to 5.9, pH 5.5 to 5.9,pH 5.4 to 5.7, pH 5.5 to 5.7, or pH 5.4 to 5.6 (e.g. pH 5.5). Unlessotherwise stated, such ranges are inclusive of the end points. Inanother embodiment, the pH is pH 5.0 to 6.2, pH 5.0 to 6.1, pH 5.2 to6.2, pH 5.2 to 6.1, pH 5.4 to 6.2, pH 5.4 to 6.1, pH 5.5 to 6.1, pH 5.4to 5.9, pH 5.5 to 5.9, pH 5.4 to 5.7, pH 5.5 to 5.7, or pH 5.4 to 5.6not including the end points.

In a further aspect of the invention, there is provided a method for thetreatment or prevention of Streptococcus pneumoniae infection in asubject in need thereof comprising administering to said subject atherapeutically effective amount of an immunogenic composition orvaccine of the invention.

In a further aspect of the invention, there is provided immunogeniccomposition or vaccine of the invention for use in the treatment orprevention of disease caused by Streptococcus pneumoniae infection.

The term “fragment” as used in this specification is a portion smallerthan the whole that is capable of eliciting a humoral and/or cellularimmune response in a host animal, e.g. human. Fragments of a protein canbe produced using techniques known in the art, e.g. recombinantly, byproteolytic digestion, or by chemical synthesis. Internal or terminalfragments of a polypeptide can be generated by removing one or morenucleotides from one end (for a terminal fragment) or both ends (for aninternal fragment) of a nucleic acid which encodes the polypeptide.Typically, fragments comprise at least 10, 20, 30, 40 or 50 contiguousamino acids of the full length sequence. Fragments may be readilymodified by adding or removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40or 50 amino acids from either or both of the N and C termini.

The term “conservative amino acid substitution” as used in thisspecification involves substitution of a native amino acid residue witha non-native residue such that there is little or no effect on the size,polarity, charge, hydrophobicity, or hydrophilicity of the amino acidresidue at that position, and without resulting in decreasedimmunogenicity. For example, these may be substitutions within thefollowing groups: valine, glycine; glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. Conservativeamino acid modifications to the sequence of a polypeptide (and thecorresponding modifications to the encoding nucleotides) may producepolypeptides having functional and chemical characteristics similar tothose of a parental polypeptide.

The term “deletion” as used in this specification is the removal of oneor more amino acid residues from the protein sequence. Typically, nomore than about from 1 to 6 residues (e.g. 1 to 4 residues) are deletedat any one site within the protein molecule.

The term “insertion” as used in this specification is the addition ofone or more non-native amino acid residues in the protein sequence.Typically, no more than about from 1 to 6 residues (e.g. 1 to 4residues) are inserted at any one site within the protein molecule.

As used herein, the term “treatment” (including variations thereof, e.g.“treat” or “treated”) means any one or more of the following: (i) theprevention of infection or re-infection, (ii) the reduction in theseverity of, or, in the elimination of symptoms, (iii) the delay inrecurrence of symptoms, and (iii) the substantial or completeelimination of the pathogen or disorder in a subject. Hence, treatmentmay be effected prophylactically (prior to infection) or therapeutically(following infection).

For the purposes of this invention, “treatment or prevention ofexacerbations of COPD” or “reduction in severity of COPD exacerbations”refers to a reduction in incidence or rate of COPD exacerbations (forinstance a reduction in rate of 0.1, 0.5, 1, 2, 5, 10, 20% or more) or areduction in severity of COPD exacerbations (e.g. airflow obstruction,chronic bronchitis, bronchiolitis or small airways disease andemphysema), for instance within a patient group immunized with theimmunogenic compositions or vaccines of the invention.

Pneumolysin

By “pneumolysin”, or “ply” or “Ply”, it is meant: native or wild-typepneumolysin from pneumococcus or recombinant pneumolysin having thesequence of native or wild-type pneumolysin. Expression and cloning ofwild-type or native pneumolysin is known in the art. See, for example,Walker et al. (Infect Immun, 55:1184-1189 (1987)), Mitchell et al.(Biochim Biophys Acta, 1007:67-72 (1989) and Mitchell et al (NAR,18:4010 (1990)). WO2010/071986 describes wild-type Ply, e.g. SEQ ID NOs2-42 (for example SEQ ID NOs 34, 35, 36, 37, 41). Furthermore,EP1601689B1 describes methods for purifying bacterial cytolysins such aspneumococcal pneumolysin by chromatography in the presence of detergentand high salt. In an embodiment, native or wild-type pneumolysin frompneumococcus or recombinant pneumolysin having the sequence of native orwild-type pneumolysin is used to generate detoxified pneumolysin. In oneaspect, pneumolysin used to generate detoxified pneumolysin has thesequence of Seq ID No. 1 (Seq ID No. 34 of WO2010/071986). In anotheraspect, pneumolysin used to generate detoxified pneumolysin has thesequence of Seq ID No. 2 (Seq ID No. 35 of WO2010/071986). In anotheraspect, pneumolysin used to generate detoxified pneumolysin has thesequence of Seq ID No. 3 (Seq ID No. 36 of WO2010/071986). In anotheraspect, pneumolysin used to generate detoxified pneumolysin has thesequence of Seq ID No. 4 (Seq ID No. 37 of WO2010/071986). In anotheraspect, pneumolysin used to generate detoxified pneumolysin has thesequence of Seq ID No. 5 (Seq ID No. 41 of WO2010/071986).

In an embodiment, the pneumolysin used to generate detoxifiedpneumolysin includes fragments and/or variants, having differences innucleic acid or amino acid sequences as compared to a wild type sequence(e.g. Seq ID Nos 1-5).

Where fragments of pneumolysin are used, these fragments will be atleast about 15, at least about 20, at least about 40, or at least about60 contiguous amino acid residues in length. In an embodiment of theinvention, immunogenic fragments of pneumolysin comprise at least about15, at least about 20, at least about 40, or at least about 60contiguous amino acid residues of the full length sequence, wherein saidpolypeptide is capable of eliciting an immune response specific for saidamino acid sequence. Native pneumolysin is known to consist of fourmajor structural domains (Rossjohn et al. Cell. 1997 May 30;89(5):685-92). These domains may be modified by removing and/ormodifying one or more of these domains. In an embodiment, the or eachfragment contains exactly or at least 1, 2 or 3 domains. In anotherembodiment, the or each fragment contains exactly or at least 2 or 3domains. In another embodiment, the or each fragment contains at least 3domains. The or each fragment may be more than 50, 60, 70, 80, 90 or100% identical to a wild type pneumolysin sequence.

In accordance with the present invention, a variant of pneumolysin is aprotein in which the native pneumolysin is mutated. The term “mutated”is used herein to mean pneumolysin which has undergone deletion and/oraddition and/or substitution of one or more amino acids (e.g. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids). Amino acid substitution maybe conservative or non-conservative. In one aspect, amino acidsubstitution is conservative. Substitutions, deletions, additions or anycombination thereof may be combined in a single variant so long as thevariant is an immunogenic polypeptide. Variants of pneumolysin typicallyinclude any pneumolysin or any fragment of pneumolysin which shares atleast 80, 90, 94, 95, 98, or 99% amino acid sequence identity with awild-type pneumolysin sequence, for example a wild-type pneumolysinsequence disclosed in WO2010/071986. In an embodiment, variants ofpneumolysin typically include any pneumolysin or any fragment ofpneumolysin which shares at least 80, 90, 94, 95, 96, 97, 98, or 99%amino acid sequence identity with SEQ ID NO: 1. In an embodiment,variants of pneumolysin typically include any pneumolysin or anyfragment of pneumolysin which shares at least 80, 90, 94, 95, 96, 97,98, or 99% amino acid sequence identity with SEQ ID NO: 2. In anembodiment, variants of pneumolysin typically include any pneumolysin orany fragment of pneumolysin which shares at least 80, 90, 94, 95, 96,97, 98, or 99% amino acid sequence identity with SEQ ID NO: 3. In anembodiment, variants of pneumolysin typically include any pneumolysin orany fragment of pneumolysin which shares at least 80, 90, 94, 95, 96,97, 98, or 99% amino acid sequence identity with SEQ ID NO: 4. In anembodiment, variants of pneumolysin typically include any pneumolysin orany fragment of pneumolysin which shares at least 80, 90, 94, 95, 96,97, 98, or 99% amino acid sequence identity with SEQ ID NO: 5. In anembodiment, the present invention includes fragments and/or variants inwhich several, 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acids aresubstituted, deleted, or added in any combination. In anotherembodiment, the present invention includes fragments and/or variantswhich comprise a B-cell or T-cell epitope. Such epitopes may bepredicted using a combination of 2D-structure prediction, e.g. using thePSIPRED program (from David Jones, Brunel Bioinformatics Group, Dept.Biological Sciences, Brunel University, Uxbridge UB8 3PH, UK) andantigenic index calculated on the basis of the method described byJameson and Wolf (CABIOS 4:181-186 [1988]). Variants of pneumolysin aredescribed for example in WO04/43376, WO05/108580, WO05/076696,WO10/071986, WO10/109325 (SEQ ID NOs 44, 45 and 46) and WO10/140119 (SEQID NOs 50 and 51). In an embodiment, the immunogenic composition of theinvention comprises a variant of pneumolysin, for example, thosedescribed in WO05/108580, WO05/076696, WO10/071986.

In an embodiment of the invention, the pneumolysin and its fragmentsand/or variants thereof, used to generate detoxified pneumolysin, havean amino acid sequence sharing at least 80, 85, 90, 95, 96, 97, 98, 99or 100% identity with the wild type sequence for pneumolysin, e.g. SEQID NOs 1, 2, 3, 4 or 5. In another embodiment of the invention,pneumolysin and its fragments and/or variants thereof, comprise at leastabout 15, at least about 20, at least about 40, or at least about 60contiguous amino acid residues of the wild type sequence forpneumolysin, e.g. of SEQ ID NOs 1, 2, 3, 4 or 5.

Because pneumolysin is a toxin, it needs to be detoxified (i.e. renderednon-toxic to a mammal, e.g. human, when provided at a dosage suitablefor protection) before it can be administered in vivo. As used herein,it is understood that the term “detoxified pneumolysin” or “dPly” refersto detoxified pneumolysin suitable for medical use (i.e. non toxic whenprovided to a mammal, e.g. human at a dosage suitable for protection).Pneumolysin may be detoxified chemically and/or genetically. Therefore,immunogenic compositions of the invention comprise detoxifiedpneumolysin (dPly).

Detoxification of pneumolysin can be conducted by chemical means, e.g.using a crosslinking agent, such as formaldehyde, glutaraldehyde and across-linking reagent containing an N-hydroxysuccinomido ester and/or amaleimide group (e.g. GMBS) or a combination of these, see for exampleEP1601689B1, WO04/081515, WO2006/032499. The pneumolysin subject tochemical detoxification may be a native or recombinant protein or aprotein that has been genetically engineered to reduce its toxicity (seebelow). Fragments and/or variants of pneumolysin may also be detoxifiedby chemical means. In an embodiment, immunogenic compositions of theinvention may comprise pneumolysin which has been chemically detoxified,e.g. by a formaldehyde treatment. For example, pneumolysin may bepurified and detoxified as described in WO2004/081515. Detoxification ofpneumolysin using formaldehyde may be carried out using formaldehyde inthe presence of L-lysine, for example by treatment of purifiedpneumolysin (ply) with 50 mM L-lysine and 0.1% formaldehyde (w/v) for 21days at 40° C.

Pneumolysin can also be genetically detoxified. Thus, the inventionencompasses pneumococcal proteins which may be, for example, mutatedproteins (as defined herein). In one embodiment, the molecule hasundergone deletion or substitution of 1-15 or any subset thereof, forexample, 10-15 amino acids. The mutated sequences may remove undesirableactivities such as membrane permeation, cell lysis, and cytolyticactivity against human erythrocytes and other cells, in order to reducethe toxicity, whilst retaining the ability to induce anti-pneumolysinprotective and/or neutralizing antibodies following administration to ahuman. Fusion proteins of pneumolysin or fragments and/or variants ofpneumolysin may also be detoxified by genetic means. For example, asdescribed herein, a mutant pneumolysin protein may be altered so that itis biologically inactive whilst still maintaining its immunogenicepitopes, see, for example, WO90/06951, Berry et al. (Infect Immun,67:981-985 (1999)) and WO99/03884. Alternatively, a pneumolysin proteinmay be detoxified by three amino acid substitutions comprising T₆₅ to C,G₂₉₃ to C and C₄₂₈ to A as described in WO2010/071986. For example, oneof SEQ ID NOs 1 to 5 could be detoxified by three amino acidsubstitutions comprising T₆₅ to C, G₂₉₃ to C and C₄₂₈ to A. Anotherexample of a genetically detoxified pneumolysin that can be used in thepresent invention is SEQ ID NO: 9 from WO2011/075823. In another aspect,the modified pneumolysin protein of the invention may be detoxified byamino acid substitutions as described in Taylor et al. PLOS ONE 8(4):e61300 (2013), for example A₃₇₀ to E, W₄₃₃ to E and/or L₄₆₀ to E. Thus,in a further embodiment, immunogenic compositions of the invention maycomprise pneumolysin which has been genetically detoxified. Acombination of techniques may also be used to detoxify pneumolysin. Forexample, immunogenic compositions of the invention may comprisepneumolysin which has been chemically and genetically detoxified.

In one aspect the detoxified pneumolysin is conjugated to a saccharide,e.g. a capsular saccharide of S. pneumoniae. For example, pneumolysinmay be conjugated to a capsular saccharide of S. pneumoniae selectedfrom serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. In a particular aspect,pneumolysin may be conjugated to capsular saccharide of S. pneumoniaeserotype 19A. In another aspect, the pneumococcal protein isunconjugated or present in the immunogenic composition as a freeprotein.

Aluminium Phosphate

Immunogenic compositions of the invention include an aluminium phosphateadjuvant. Aluminium phosphate (including both anhydrous and hydratedforms) is often referred to for convenience as “AlPO₄”, althoughhydrated forms (hydroxyphosphates) can be distinguished from anhydrousAlPO₄ by the presence of hydroxyl groups (Al(OH)_(x)(PO₄)_(y), e.g.Al(OH)(PO₄)). In one aspect of the invention, the aluminium phosphate isaluminium hydroxyphosphate (e.g. amorphous aluminium hydroxyphosphate).In another aspect of the invention, the aluminium phosphate is aluminiumorthophosphate (also known as “aluminium monophopshate”). Aluminiumphosphate adjuvants may be purchased from Brenntag, e.g. aluminiumphosphate gel adjuvant.

Aluminium phosphate can be a precipitate of insoluble aluminiumphosphate (amorphous, semi-crystalline or crystalline) which may beprepared by mixing soluble aluminium salts and phosphoric acid salts,e.g. sodium phosphate or potassium phosphate. In one aspect, thealuminium phosphate is amorphous (e.g. amorphous hydroxyphosphate).Aluminium hydroxyphosphate is not a stoichiometric compound and itshydroxyl and phosphate composition depends on precipitation reactantsand conditions. The Phosphate:Aluminium (P:Al) weight/weight (w/w) of analuminium hydroxyphosphate adjuvant will generally be between 2:1 to4:1, suitably between 2.5:1 to 3.5:1, or between 3:1 to 3.5:1. Thealuminium content may be determined by atomic absorptionspectrophotometry with nitrous flame, see for example May et al. (1984)J. Biol. Stand. 12(2):175-83.

In one embodiment, the aluminium phosphate used in the process of theinvention comprises NaCl, suitably 0.8% to 1.0%, e.g. 0.9% (w/w).

In an embodiment, the aluminium phosphate used in the process of theinvention has a pH between 4.8 and 6.2. In another embodiment, thealuminium phosphate used in the process of the invention has a pHbetween 5.5 and 6.1. In another embodiment, the aluminium phosphate usedin the process of the invention has a pH between 4.8 and 5.8. In anotherembodiment, the aluminium phosphate used in the process of the inventionhas a pH between 5.2 and 5.8.

In one embodiment, the aluminium phosphate used in the process of theinvention is “extra-washed” prior to the adsorption of dPly such thatthe free phosphate ion concentration is reduced to below 10 mM (e.g. 3mM or less, 2.5 mM or less). For example, the phosphate ions may beremoved either by repeated centrifugation (e.g. at least 3 times) anddilution steps (i.e. removal of the supernatant and resuspension of thepellet in saline), or by diafiltration steps.

In another embodiment, the aluminium phosphate should be sterilisedbefore adsorption of antigen. In one aspect, the aluminium phosphate issterilised by autoclaving. In another aspect the aluminium phosphate issterilised by irradiation, e.g. using ultra violet (UV) light.

Completeness of adsorption of a protein antigen (e.g. dPly) ontoaluminium phosphate can be measured by measuring the supernatant (SN) ofcentrifuged samples via Lowry and comparing the total amount of proteinin the sample (measured before adsorption occurs or by desorbingadsorbed antigen) to the amount which remains in the supernatant aftercentrifugation, as described in Example 2 herein. This methodology isfurther described in Chapter 4 of Methods in Molecular Medicine, Vol. 42(edited by D.T. O-Hagan) Vaccine Adjuvants Preparation Methods andResearch Protocols. In one aspect, the present invention providesdetoxified pneumolysin adsorbed onto aluminium phosphate, wherein morethan 85% (suitably more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%) of the dPly is adsorbed onto the aluminium phosphate. In oneaspect of the invention, completeness of adsorption is measured on theday of formulation (T0). In another aspect of the invention,completeness of adsorption is measured after 7 days at +4° C. (T7d4° C.)following formulation. In another aspect of the invention, completenessof adsorption is measured after 21 days at +4° C. (T21d4° C.) followingformulation. In another aspect of the invention, completeness ofadsorption is measured after 7 days under accelerated conditions, e.g. 7days at 37° C. (7d37° C.) following formulation. In another aspect ofthe invention, completeness of adsorption is measured after 16 daysunder accelerated conditions, e.g. 10 days at 4° C. followed by 6 daysat 37° C. (T10d4° C.+6d37° C.) following formulation.

Particle size of a protein antigen (e.g. dPly) adsorbed onto aluminiumphosphate, can be measured by SLS (static light scattering), forexample, using a Hydro 2000 μP dispersant unit as described in Example 4herein (methods for determining particle size are further described inE. Lindblad, Immunology and Cell Biology (2004) 82: 497-505). Thescattering intensity is a function of the molecular weight andconcentration. In one aspect, the present invention provides detoxifiedpneumolysin adsorbed onto aluminium phosphate, wherein greater than 80%(suitably more than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%)of the particles of detoxified pneumolysin adsorbed onto aluminiumphosphate have a size less than 10 μm. In one aspect, the detoxifiedpneumolysin is unconjugated detoxified pneumolysin. In another aspect,the detoxified pneumolysin is conjugated detoxified pneumolysin.

Process for Adsorption

The present invention provides a process for adsorption of detoxifiedpneumolysin onto aluminium phosphate comprising the step of (i) admixingdetoxified pneumolysin and the aluminium phosphate at a pH less than 6.5(e.g. less than 6.4, less than 6.3, less than 6.2, less than 6.1),suitably less than pH 6.0, for example pH 5.0 to 6.2, pH 5.0 to 6.1, pH5.2 to 6.2, pH 5.2 to 6.1, pH 5.4 to 6.2, pH 5.4 to 6.1, pH 5.5 to 6.1,pH 5.4 to 5.9, pH 5.5 to 5.9, pH 5.4 to 5.7, pH 5.5 to 5.7, or pH 5.4 to5.6 (e.g. pH 5.5). Suitably, the ratio of dPly:Al³⁺ (from aluminiumphosphate) in step (i) is between 1:1.5 to 1:5.5, between 1:1.5 to 1:4,between 1:1.5 to 1:3.5, between 1:1.5 to 1:2.5, between 1:2 to 1:2.5(e.g. 1:2); or between 1:2.5 to 1:3.5, between 1:3 to 1:3.5 (e.g. 1:3),or between 1:3 to 1:4 (e.g. 1:3.5) (w/w; weight/weight). In one aspect,dPly is adsorbed onto aluminium phosphate in a ratio of 1 μg of dPly to3 μg of Al³⁺ (from aluminium phosphate). In one embodiment, the pH is5.4 to 6.2 (e.g. pH5.5+/−0.1) and the ratio of dPly:Al³⁺ is between1:2.5 to 1:3.5 (e.g. 1:3) (w/w; weight/weight). In another embodiment,the pH is 5.4 to 6.2 (e.g. pH6.1+/−0.1) and the ratio of dPly:Al³⁺ isbetween 1:1.5 to 1:3.5 (e.g. 1:2.5) (w/w; weight/weight). In anotherembodiment, the pH is 5.4 to 6.2 (e.g. pH6.1+/−0.1) and the ratio ofdPly:Al³⁺ is between 1:3 to 1:4 (e.g. 1:3.5) (w/w; weight/weight).Unless otherwise stated, such ranges are inclusive of the end points. Inanother embodiment, the ratio of dPly:Al³⁺ (from aluminium phosphate) instep (i) is between 1:1.5 to 1:5.5, between 1:1.5 to 1:4, between 1:1.5to 1:3.5, between 1:1.5 to 1:2.5, between 1:2 to 1:2.5; or between 1:2.5to 1:3.5, between 1:3 to 1:3.5, or between 1:3 to 1:4 (w/w;weight/weight) not including the end points.

Suitably, for conjugated detoxified pneumolysin, the ratio ofpolysaccharide:Al³⁺ (from aluminium phosphate) in step (i) is between1:6 to 1:14, between 1:7 to 1:13, between 1:7.5 to 1:12.5, between 1:8to 1:12 (e.g. 1:10) (w/w; weight/weight). In another aspect, conjugateddPly is adsorbed onto aluminium phosphate in a ratio of 1 μg ofpolysaccharide to 10 μg of Al³⁺ (from aluminium phosphate). In oneembodiment, the pH is 5.4 to 6.2 (e.g. pH6.1+/−0.1) and the ratio ofpolysaccharide:Al³⁺ is between 1:7.5 to 1:12.5 (e.g. 1:10) (w/w;weight/weight).

Suitably, step (i) is carried out at room temperature (18-24° C.).Suitably, step (i) is carried out with stirring at between 60 to 150rpm, such as 120 to 140 rpm (e.g. 130 rpm). Suitably, step (i) iscarried out for between 10 minutes to 2 weeks, for example, 10 minutesto 5 hours, 1 to 5 hours, or 2 to 3 hours. Unless otherwise stated, suchranges are inclusive of the end points. In another embodiment, the pH ismaintained (and the further mixing continues) at this pH (i.e. the pHfor adsorption) for between 10 minutes to 2 weeks, for example, 10minutes to 5 hours, 1 to 5 hours, or 2 to 3 hours not including the endpoints. In an embodiment of step (i), the detoxified pneumolysin and thealuminium phosphate (and optionally a buffer) are initially mixed andsubsequently (e.g. after 5-15 minutes) the pH is adjusted to a pH lessthan 6.5 (e.g. less than 6.4, less than 6.3, less than 6.2, less than6.1), suitably less than pH 6.0, for example pH 5.0 to 6.2, pH 5.0 to6.1, pH 5.2 to 6.2, pH 5.2 to 6.1, pH 5.4 to 6.2, pH 5.4 to 6.1, pH 5.5to 6.1, pH 5.4 to 5.9, pH 5.5 to 5.9, pH 5.4 to 5.7, pH 5.5 to 5.7, orpH 5.4 to 5.6 (e.g. pH 5.5) (the pH for adsorption) followed by furthermixing. The pH may be adjusted using sodium hydroxide (NaOH (aq)) andhydrochloric acid (HCl (aq)). Suitably, the pH is maintained (and thefurther mixing continues) at this pH (i.e. the pH for adsorption) forbetween 10 minutes to 2 weeks, for example, 10 minutes to 5 hours, 1 to5 hours, or 2 to 3 hours. Unless otherwise stated, such ranges areinclusive of the end points. In another embodiment, the pH is maintained(and the further mixing continues) at this pH (i.e. the pH foradsorption) for between 10 minutes to 2 weeks, for example, 10 minutesto 5 hours 1 to 5 hours, or 2 to 3 hours not including the end points.

In an aspect of the invention, the process of the invention (i.e.adsorption of dPly, step (i)), is carried out in the presence of abuffer, such as phosphate buffer (e.g. NaK₂). In one aspect, theconcentration of the buffer (e.g. NaK₂) is at least 1 mM (e.g. at least1.5 mM, 2 mM, 2.3 mM, 3 mM, 4 mM) and is suitably at most 10 mM (e.g. atmost 9 mM, 8 mM, 7 mM, 6 mM, 5 mM). In another aspect the concentrationof the buffer (e.g. NaK₂) is between 1 mM and 5 mM, or between 1 and 4mM, or between 1 mM and 3 mM (e.g. between 2 mM and 3 mM), for example,between 2 mM and 2.4 mM, e.g. 2 mM. The phosphate buffer, NaK₂, used inthe adsorption of dPly may comprise (sodium phosphate monobasic) NaH₂PO₄and (potassium phosphate dibasic) K₂HPO₄. Suitably, the buffer has a pH6.5 to 7.5 (e.g. pH 7.15). Unless otherwise stated, such ranges areinclusive of the end points. In another embodiment, the concentration ofthe buffer (e.g. NaK₂) is between 1 mM and 5 mM, or between 1 and 4 mM,or between 1 mM and 3 mM (e.g. between 2 mM and 3 mM) not including theend points.

In an aspect of the invention, the process of the invention (i.e.adsorption of dPly, step (i)), is carried out in the presence of asodium salt, e.g. NaCl. In one aspect, the concentration of the sodiumsalt is between 20 to 160 mM, 30 to 150 mM, 40 to 65 mM, 45 to 65 mM, 50to 60 mM (e.g. 55 mM). Unless otherwise stated, such ranges areinclusive of the end points. In another embodiment, the concentration ofthe sodium salt is between 20 to 160 mM, 30 to 150 mM, 40 to 65 mM, 45to 65 mM, 50 to 60 mM not including the end points.

In an aspect of the invention, the process further comprises the step(ii) adjustment of the pH of the composition to a pH between 6 and 7(for example pH 6.0 to 6.5, pH 6.0 to 6.3, pH 6.1). Step (ii) issuitably carried out following step (i). Suitably, step (i) is carriedout at room temperature (18-24° C.). Suitably, step (i) is carried outwith stirring at between 60 to 150 rpm (e.g. 130 rpm). The pH may beadjusted using NaOH and HCl.

Following step (i) and (ii), suitably the adsorbed dPly is maintained ata pH between 6 and 7 (for example pH 6.0 to 6.5, pH 6.0 to 6.3, pH 6.1)for at least 7 days, suitably at 2-8° C. (maturation step). Accordingly,immunogenic compositions of the invention may have a pH between 6 and 7(for example pH 6.0 to 6.5, pH 6.0 to 6.3, pH 6.1).

In one embodiment, the adsorption of dPly onto aluminium phosphate (withor without sodium salt or potassium salt) is carried out in the absenceof other additives, for example in the absence of histidine.

The present invention also provides a process for preparing animmunogenic composition of the invention comprising the process of theinvention for adsorption of detoxified pneumolysin onto aluminiumphosphate as described herein.

Immunogenic Compositions

In an embodiment, the present invention provides an immunogeniccomposition comprising detoxified pneumolysin adsorbed onto aluminiumphosphate prepared by the process of the invention.

In one aspect, the present invention provides an immunogenic compositioncomprising detoxified pneumolysin adsorbed onto aluminium phosphate,wherein more than 85% (suitably more than 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99%) of the detoxified pneumolysin is adsorbed onto thealuminium phosphate. In another aspect, the present invention providesan immunogenic composition wherein greater than 80% (suitably more than81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%) of the particles ofdetoxified pneumolysin adsorbed onto aluminium phosphate have a sizeless than 10 μm. In an embodiment, the pH of the immunogenic compositionis between pH6 and pH7 (for example pH 6.0 to 6.5, pH 6.0 to 6.2, pH6.1). Immunogenic compositions may be buffered at this pH, e.g. using aphosphate buffer. In one aspect, the detoxified pneumolysin in theimmunogenic composition is unconjugated detoxified pneumolysin. Inanother aspect, the detoxified pneumolysin in the immunogenic isconjugated detoxified pneumolysin.

The immunogenic composition of the invention (i.e. comprising adsorbeddPly), may also comprise a buffer, such as phosphate buffer (e.g. NaK₂).In one aspect, the concentration of the buffer (e.g. NaK₂) is at least 1mM (e.g. at least 1.5 mM, 2 mM, 2.3 mM, 3 mM, 4 mM) and is suitably atmost 10 mM (e.g. at most 9 mM, 8 mM, 7 mM, 6 mM, 5 mM). In anotheraspect the concentration of the buffer (e.g. NaK₂) is between 1 mM and 5mM, or between 1 mM and 3 mM (e.g. between 2 mM and 3 mM), for example,between 2 mM and 2.4 mM. The phosphate buffer, NaK₂, used in theadsorption of dPly may comprise (sodium phosphate monobasic) NaH₂ PO₄and (potassium phosphate dibasic) K₂HPO₄. Other buffers that could beused include histidine, sodium phosphate, potassium phosphate,carbonate, NaHCO₃ buffers. Other buffers that could be used also includemaleate, succinate, tartrate and Tris-Maleate buffers.

In an aspect of the invention, the immunogenic composition of theinvention (i.e. comprising adsorbed dPly), may also comprise a sodiumsalt, e.g. NaCl. In one aspect, the concentration of the sodium salt isbetween 20 to 160 mM, 30 to 150 mM, 40 to 65 mM, 45 to 65 mM, 50 to 60mM (e.g. 55 mM). In another aspect, the concentration of the sodium saltis between 100 to 200 mM, 120 to 180 mM, 140 to 160 mM (e.g. 150 mM).Unless otherwise stated, such ranges are inclusive of the end points. Inanother embodiment, the concentration of the sodium salt is between 20to 160 mM, 30 to 150 mM, 40 to 65 mM, 45 to 65 mM, 50 to 60 mM notincluding the end points.

In a further aspect, the immunogenic composition comprises less than2mgAl³⁺/ml, suitably between 100-2000 μgAl³⁺/ml, 500-2000 μgAl³⁺/ml,800-2000 μgAl³⁺/ml, 800-1500 μgAl³⁺/ml, 800-1200 μgAl³⁺/ml, 1000-2000μgAl³⁺/ml, 1500-2000 μgAl³⁺/ml or 1700-2000 μgAl³⁺/ml (aluminium, Al³⁺)as aluminium phosphate. Unless otherwise stated, such ranges areinclusive of the end points. In another embodiment, the immunogeniccomposition comprises between 100-2000 μgAl³⁺/ml, 500-2000 μgAl³⁺/ml,800-2000 μgAl³⁺/ml, 800-1500 μgAl³⁺/ml, 800-1200 μgAl³⁺/ml, 1000-2000μgAl³⁺/ml, 1500-2000 μgAl³⁺/ml or 1700-2000 μgAl³⁺/ml (aluminium, Al³⁺)as aluminium phosphate, not including the end points.

In a further aspect, the immunogenic composition comprises water forinjection (WFI).

Immunogenic compositions of the invention may be lyophilised or inaqueous form, i.e. solutions or suspensions. Immunogenic compositions ofthe invention may be lyophilised in the presence of a stabilisingexcipient such as sucrose or trehalose. Immunogenic compositions may bepresented in vials, or they may be presented in ready filled syringes.

The present invention also provides a process for preparing animmunogenic composition comprising detoxified pneumolysin, comprisingthe process of the invention.

Additional Antigens

Immunogenic compositions of the present invention may compriseadditional antigens capable of eliciting an immune response against ahuman or animal pathogen. These additional antigens include, forexample, additional S. pneumoniae antigens, e.g. S. pneumoniae proteinantigens. Such proteins may be used as carrier proteins, or may bepresent as a free protein (unconjugated), or may be present both as acarrier protein and a free protein. Where the additional antigen is apneumococcal protein, the protein may be conjugated for example to asaccharide. In an embodiment, the immunogenic composition of theinvention further comprises one or more unconjugated S. pneumoniaeproteins, for example, unconjugated pneumococcal polyhistidine triadprotein D (PhtD). In another embodiment, the immunogenic composition ofthe invention further comprises one or more conjugated S. pneumoniaeproteins, for example, conjugated pneumococcal polyhistidine triadprotein D (PhtD).

The additional Streptococcus pneumoniae antigens are either surfaceexposed, at least during part of the life cycle of the pneumococcus, orare proteins which are secreted or released by the pneumococcus. In anembodiment, the S. pneumoniae antigens are selected from the followingcategories, such as proteins having a Type II Signal sequence motif ofLXXC (where X is any amino acid, e.g. the polyhistidine triad family(PhtX)), choline binding proteins (e.g. CbpX (choline binding proteinfamily), PcpA (pneumococcal choline-binding protein A)), proteins havinga Type I Signal sequence motif (e.g. Sp101), and proteins having a LPXTGmotif (where X is any amino acid, e.g., Sp128, Sp130). Preferredexamples within these categories (or motifs) are the following proteins,or immunologically functional equivalents thereof. Thus, the immunogeniccomposition of the invention may comprise one or more S. pneumoniaeproteins selected from polyhistidine triad family (PhtX), CholineBinding Protein family (CbpX), CbpX truncates, pneumococcal autolysinfamily (LytX) (e.g. LytA (N-acetylmuramoyl-l-alanine amidase), LytB,LytC), LytX truncates, CbpX truncate-LytX truncate chimeric proteins,PspA (pneumococcal surface protein A), PsaA (pneumococcal surfaceadhesion A), Sp128, Sp101, Sp130, Sp125 and Sp133. In a furtherembodiment, the immunogenic composition of the invention comprises 2 ormore proteins selected from the group consisting of the polyhistidinetriad family (PhtX), Choline Binding Protein family (CbpX), CbpXtruncates, LytX family, LytX truncates, CbpXtruncate-LytXtruncatechimeric proteins (or fusions), PspA (pneumococcal surface protein A),PsaA (pneumococcal surface adhesion A), and Sp128. In a furtherembodiment, the immunogenic composition comprises 2 or more proteinsselected from the group consisting of the polyhistidine triad family(PhtX), Choline Binding Protein family (CbpX), CbpX truncates, LytXfamily, LytX truncates, CbpX truncate-LytX truncate chimeric proteins(or fusions), and Sp128.

The Pht (polyhistidine triad) family comprises proteins PhtA, PhtB,PhtD, and PhtE. The family is characterized by a lipidation sequence,two domains separated by a proline-rich region and several histidinetriads, possibly involved in metal or nucleoside binding or enzymaticactivity, (3-5) coiled-coil regions, a conserved N-terminus and aheterogeneous C terminus. It is present in all strains of pneumococcitested. Homologous proteins have also been found in other Streptococciand Neisseria. In one embodiment of the invention, the immunogeniccomposition comprises PhtD. It is understood, however, that the termsPht A, B, D, and E refer to proteins having sequences disclosed in thecitations below as well as variants thereof that have a sequencehomology that is at least 90% identical to the proteins described below,e.g. amino acids 21-838 of SEQ ID NO: 4 of WO00/37105. In an embodimentit is at least 95% identical and in another embodiment it is 97%identical to the proteins described below, e.g. amino acids 21-838 ofSEQ ID NO: 4 of WO00/37105.

With regards to the PhtX proteins, PhtA is disclosed in WO 98/18930, andis also referred to Sp36. As noted herein, it is a protein from thepolyhistidine triad family and has the type II signal motif of LXXC.PhtD is disclosed in WO 00/37105, and is also referred to Sp036D. Asnoted herein, it also is a protein from the polyhistidine triad familyand has the type II LXXC signal motif. PhtB is disclosed in WO 00/37105,and is also referred to Sp036B. Another member of the PhtB family is theC3-Degrading Polypeptide, as disclosed in WO 00/17370. This protein alsois from the polyhistidine triad family and has the type II LXXC signalmotif. A preferred immunologically functional equivalent is the proteinSp42 disclosed in WO 98/18930. A PhtB truncate (a “truncate” being partof a protein having an N-terminal and/or C-terminal deletion)(approximately 79 kD) is disclosed in WO99/15675 which is alsoconsidered a member of the PhtX family. PhtE is disclosed in WO00/30299and is referred to as BVH-3. Where any Pht protein is referred toherein, it is meant that immunogenic fragments or fusions thereof of thePht protein can be used.

In one embodiment, the S. pneumoniae antigen selected from member(s) ofthe polyhistidine triad family is PhtD. The term “PhtD” as used hereinincludes the full length protein with the signal sequence attached orthe mature full length protein with the signal peptide (for example 20amino acids at N-terminus) removed, and immunogenic fragments, variantsand/or fusion proteins thereof, e.g. SEQ ID NO: 4 of WO00/37105. In oneaspect, PhtD is the full length protein with the signal sequenceattached e.g. SEQ ID NO: 4 of WO00/37105. In another aspect, PhtD is asequence comprising the mature full length protein with the signalpeptide (for example 20 amino acids at N-terminus) removed, e.g. aminoacids 21-838 of SEQ ID NO: 4 of WO00/37105. Suitably, the PhtD sequencecomprises an N-terminal methionine. The present invention also includesPhtD polypeptides which are immunogenic fragments of PhtD, variants ofPhtD and/or fusion proteins of PhtD. For example, as described inWO00/37105, WO00/39299, U.S. Pat. No. 6,699,703 and WO09/12588.

Where immunogenic fragments of PhtD proteins are used (separately or aspart of a fusion protein), these immunogenic fragments will be at leastabout 15, at least about 20, at least about 40, or at least about 60contiguous amino acid residues in length, e.g. from a PhtD amino acidsequence in WO00/37105 or WO00/39299, such as SEQ ID NO: 4 ofWO00/37105. In an embodiment of the invention, immunogenic fragments ofPhtD protein comprise at least about 15, at least about 20, at leastabout 40, or at least about 60 contiguous amino acid residues of thesequence shown in SEQ ID NO: 4 of WO00/37105, wherein said polypeptideis capable of eliciting an immune response specific for said amino acidsequence. In an embodiment, the immunogenic composition of the inventioncomprises an immunogenic fragment of PhtD, for example described inWO09/12601, WO01/98334 and WO09/12588. Where immunogenic fragments ofPhtD proteins are used (separately or as part of a fusion protein), eachimmunogenic fragment optionally contains one or more histidine triadmotif(s) of such polypeptides. A histidine triad motif is the portion ofpolypeptide that has the sequence HxxHxH where H is histidine and x isan amino acid other than histidine. In an embodiment of the presentinvention, the or each immunogenic fragment contains exactly or at least2, 3, 4 or 5 histidine triad motifs (optionally, with native PhtDsequence between the 2 or more triads, or intra-triad sequence) wherethe immunogenic fragment is more than 50, 60, 70, 80, 90 or 100%identical to a native pneumococcal intra-triad PhtD sequence (e.g. theintra-triad sequence shown in SEQ ID NO: 4 of WO00/37105). Immunogenicfragments of PhtD proteins optionally contain one or more coiled coilregions of such polypeptides. A coiled coil region is a region predictedby “Coils” algorithm Lupus, A et al (1991) Science 252; 1162-1164. In anembodiment of the present invention, each immunogenic fragment containsexactly or at least 2, 3 or 4 coiled coil regions. In an embodiment ofthe present invention, the or each immunogenic fragment contains exactlyor at least 2, 3 or 4 coiled coil regions where the immunogenic fragmentis more than 50, 60, 70, 80, 90, 95, 96 or 100% identical to a nativepneumococcal PhtD sequence (e.g. the sequence shown in SEQ ID NO: 4 ofWO00/37105). In another embodiment of the present invention, theimmunogenic fragment includes one or more histidine triad motif as wellas at least 1, 2, 3 or 4 coiled coil regions.

In the case where the PhtD polypeptide is a variant, the variation isgenerally in a portion thereof other than the histidine triad residuesand the coiled-coil region, although variations in one or more of theseregions may be made. In accordance with the present invention, a variantis a protein in which the native pneumolysin is mutated. Amino acidsubstitution may be conservative or non-conservative. In one aspect,amino acid substitution is conservative. Substitutions, deletions,insertions or any combination thereof may be combined in a singlevariant so long as the variant is an immunogenic polypeptide. Variantstypically include polypeptides which share at least 80, 90, 94, 95, 98,or 99% amino acid sequence identity with a wild-type sequence. Variantsof PhtD typically include any immunogenic fragment or variation of PhtDwhich shares at least 80, 90, 95, 96, 98, or 99% amino acid sequenceidentity with a wild-type PhtD sequence, e.g. SEQ ID NO: 4 ofWO00/37105. In an embodiment, the present invention includes immunogenicfragments and/or variants in which several, 5 to 10, 1 to 5, 1 to 3, 1to 2 or 1 amino acid(s) are substituted, deleted, or added in anycombination. In another embodiment, the present invention includesimmunogenic fragments and/or variants which comprise a B-cell or T-cellepitope. Such epitopes may be predicted using a combination of2D-structure prediction, e.g. using the PSIPRED program (from DavidJones, Brunel Bioinformatics Group, Dept. Biological Sciences, BrunelUniversity, Uxbridge UB8 3PH, UK) and antigenic index calculated on thebasis of the method described by Jameson and Wolf (CABIOS 4:181-186[1988]).

In an embodiment of the invention, PhtD and its immunogenic fragments,variants and/or fusion proteins thereof comprise an amino acid sequencesharing at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity withamino acid sequence 21 to 838 of SEQ ID NO:4 of WO00/37105. In anotherembodiment of the invention, PhtD and its immunogenic fragments,variants and/or fusion proteins thereof have an amino acid sequencesharing at least 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity withamino acid sequence 21 to 838 of SEQ ID NO:4 of WO00/37105. Suitably,PhtD and its immunogenic fragments, variants and/or fusion proteinsthereof comprise an amino acid sequence having an N-terminal methionine.In another embodiment of the invention, PhtD and its immunogenicfragments, variants and/or fusion proteins thereof comprise at leastabout 15, at least about 20, at least about 40, or at least about 60 orat least about 100, or at least about 200, or at least about 400 or atleast about 800 contiguous amino acid residues of the sequence shown inSEQ ID NO: 4 of WO00/37105.

In one aspect the PhtD is conjugated to a saccharide, e.g. a capsularsaccharide of S. pneumoniae. For example, PhtD may be conjugated to acapsular saccharide of S. pneumoniae selected from serotypes 1, 2, 3, 4,5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15, 17F, 18C, 19A, 19F, 20,22F, 23F and 33F. In particular, PhtD may be conjugated to a capsularsaccharide of S. pneumoniae serotype 22F. In another aspect, PhtD isunconjugated or present in the immunogenic composition as a freeprotein. In an aspect of the invention, more than 80% (e.g. more than82%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of thePhtD is adsorbed onto aluminium phosphate. In another aspect of theinvention, greater than 80% (e.g. more than 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89% or 90%) of the particles of PhtD (e.g. unconjugatedPhtD) adsorbed onto aluminium phosphate have a size less than 10 μm.

The present invention also provides an immunogenic compositioncomprising PhtD adsorbed onto aluminium phosphate, wherein more than 85%(e.g. more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) ofthe PhtD is adsorbed onto aluminium phosphate. The present inventionalso provides an immunogenic composition wherein greater than 80% (e.g.more than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%) of theparticles of PhtD adsorbed onto aluminium phosphate have has a particlea size less than 10 μm.

Concerning the Choline Binding Protein family (CbpX), members of thatfamily were originally identified as pneumococcal proteins that could bepurified by choline-affinity chromatography. All of the choline-bindingproteins are non-covalently bound to phosphorylcholine moieties of cellwall teichoic acid and membrane-associated lipoteichoic acid.Structurally, they have several regions in common over the entirefamily, although the exact nature of the proteins (amino acid sequence,length, etc.) can vary. In general, choline binding proteins comprise anN terminal region (N), conserved repeat regions, a proline rich region(P) and a conserved choline binding region (C), made up of multiplerepeats, that comprises approximately one half of the protein. As usedin this application, the term “Choline Binding Protein family (CbpX)” isselected from the group consisting of Choline Binding Proteins asidentified in WO97/41151, Choline binding protein A, CbpA (also referredto as PbcA (C3-binding protein A), SpsA (Streptococcus pneumoniaesecretory IgA binding protein), PspC (pneumococcal surface protein C)),Choline binding protein D (CbpD), and Choline binding protein G (CbpG).CbpA is disclosed in WO97/41151. CbpD and CbpG are disclosed inWO00/29434. PspC is disclosed in WO97/09994. PbcA is disclosed inWO98/21337. SpsA is a Choline binding protein disclosed in WO 98/39450.In an embodiment, the Choline Binding Proteins is CbpA. Another CholineBinding Protein is pneumococcal choline-binding protein A (PcpA)(Sanchez-Beato et al FEMS Microbiology Letters 164 (1998) 207-214).

Another preferred embodiment is CbpX truncates wherein “CbpX” is CbpA,CbpD or CbpG and “CbpX truncates” refers to CbpX proteins lacking 50% ormore of the Choline binding region (C). Another preferred embodiment isPcpA truncates wherein “PcpA truncates” refers to PcpA proteins lacking50% or more of the Choline binding region (C). In an embodiment, CbpXtruncates or PcpA truncates lack the entire choline binding region. Inanother embodiment, the CbpX truncates or PcpA truncates lack (i) thecholine binding region and (ii) a portion of the N-terminal half of theprotein as well, yet retain at least one repeat region. In anotherembodiment, the truncate has at least 2 repeat regions. Examples of suchpreferred embodiments are illustrated in WO99/51266 or WO99/51188,however, other choline binding proteins lacking a similar cholinebinding region are also contemplated within the scope of this invention.

The LytX family is membrane associated proteins associated with celllysis. The N-terminal domain comprises choline binding domain(s),however the LytX family does not have all the features found in the CbpAfamily noted herein and thus for the present invention, the LytX familyis considered distinct from the CbpX family. In contrast with the CbpXfamily, the C-terminal domain contains the catalytic domain of the LytXprotein family. The family comprises LytA, LytB and LytC. With regardsto the LytX family, LytA is disclosed in Ronda et al., Eur J Biochem,164:621-624 (1987). LytB is disclosed in WO 98/18930, and is alsoreferred to as Sp46. LytC is also disclosed in WO 98/18930, and is alsoreferred to as Sp91. A preferred member of that family is LytC.

Another preferred embodiment are LytX truncates wherein “LytX” is LytA,LytB or LytC and “LytX truncates” refers to LytX proteins lacking 50% ormore of the Choline binding region. Suitably such proteins lack theentire choline binding region. Yet another preferred embodiment of thisinvention are CbpX truncate-LytX truncate chimeric proteins (orfusions). In an embodiment, the CbpX truncate-LytX truncate chimericprotein comprises the repeat regions of CbpX and the C-terminal portion(Cterm, i.e., lacking the choline binding domains) of LytX (e.g.,LytCCterm or Sp91Cterm). In another embodiment, CbpX is selected fromthe group consisting of CbpA, PbcA, SpsA and PspC. In anotherembodiment, it is CbpA. In an embodiment, LytX is LytC (also referred toas Sp91). Another embodiment of the present invention is a PspA(pneumococcal surface protein A) or PsaA (pneumococcal surface adhesionA) truncates lacking the choline binding domain (C) and expressed as afusion protein with LytX. In an embodiment, LytX is LytC.

PsaA (pneumococcal surface adhesion A) and transmembrane deletionvariants thereof have been described by Berry & Paton, Infect Immun 1996December; 64(12):5255-62. PspA (pneumococcal surface protein A) andtransmembrane deletion variants thereof have been disclosed in, forexample, U.S. Pat. No. 5,804,193, WO 92/14488, and WO 99/53940.

Sp128 and Sp130 are disclosed in WO00/76540. Sp125 is an example of apneumococcal surface protein with the Cell Wall Anchored motif of LPXTG(i.e. leucine-proline-X-threonine-glycine where X is any amino acid).Any protein within this class of pneumococcal surface protein with thismotif has been found to be useful within the context of this invention,and is therefore considered a further protein of the invention. Sp125itself is disclosed in WO 98/18930, and is also known as ZmpB—a zincmetalloproteinase. Sp101 is disclosed in WO 98/06734 (where it has thereference #y85993). It is characterized by a Type I signal sequence.Sp133 is disclosed in WO 98/06734 (where it has the reference #y85992).It is also characterized by a Type I signal sequence.

The S. pneumoniae antigens may also be beneficially combined. Bycombined is meant that the immunogenic composition comprises all of theproteins from within the combination, either as carrier proteins or asfree proteins or a mixture of the two. For example, in a combination oftwo proteins as set out hereinafter, both proteins may be used ascarrier proteins, or both proteins may be present as free proteins, orboth may be present as carrier and as free protein, or one may bepresent as a carrier protein and a free protein whilst the other ispresent only as a carrier protein or only as a free protein, or one maybe present as a carrier protein and the other as a free protein. Where acombination of three proteins is given, similar possibilities exist.Preferred combinations include, but are not limited to PhtD+CbpX repeatregions, PhtD+dPly, PhtD+Sp128, PhtD+PsaA, PhtD+PspA, PhtA+CbpX repeatregions, PhtA+CbpX repeat regions -Sp91Cterm chimeric or fusionproteins, PhtA+dPly, PhtA+Sp128, PhtA+PsaA, PhtA+PspA, CbpX repeatregions+LytC, CbpX repeat regions+PspA, CbpX repeat regions+PsaA, CbpXrepeat regions+Sp128, CbpX repeat regions+LytC, CbpX repeatregions+PspA, CbpX repeat regions+PsaA, CbpX repeat regions+Sp128, CbpXrepeat regions+PhtD, CbpX repeat regions+PhtA. In an embodiment, CbpXrepeat regions is from CbpA. In another embodiment, it is from CbpA.Other combinations include 3 protein combinations such as PhtD+CbpXrepeat regions+dPly, and PhtA+CbpX repeat regions+PhtD. In oneembodiment, the immunogenic composition comprises detoxified pneumolysinand PhtD as carrier proteins. In a further embodiment, the immunogeniccomposition comprises detoxified pneumolysin and PhtD as free proteins.

The immunogenic compositions of the invention may also comprise S.pneumoniae capsular saccharides (suitably conjugated to a carrierprotein), for example as described in WO2007/071707A2. The bacterialcapsular saccharide from Streptococcus pneumoniae may be selected from aStreptococcus pneumoniae serotype 1, 2, 3, 4, 5, 6A, 6B, 7A, 7B, 7C, 8,9A, 9L, 9N, 9V, 10A, 10B, 100, 10F, 11A, 11B, 11C, 11D, 11F, 12A, 12B,12F, 13, 14, 15A, 15B, 15C, 15F, 16A, 16F, 17A, 17F, 18A, 18B, 18C, 18F,19A, 19B, 19C, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25A,25F, 26, 27, 28A, 28F, 29, 31, 32A, 32F, 33A, 33B, 33C, 33D, 33F, 34,35A, 35B, 35C, 35D, 35F, 36, 37, 38, 39, 40, 41A, 41F, 42, 43, 44, 45,46, 47A, 47F or 48 capsular saccharide. The saccharides (e.g.polysaccharides (PS)) may be derived from serotypes of pneumococcus suchas serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14,15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F. In an embodiment, atleast four serotypes are included in the composition, e.g. 6B, 14, 19Fand 23F (suitably conjugated to a carrier protein). In anotherembodiment, at least 7 serotypes are included in the composition, e.g.4, 6B, 9V, 14, 18C, 19F and 23F (suitably conjugated to a carrierprotein). In another embodiment the immunogenic composition comprises 10or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16or more, 17 or more, 19 or more, or 20 capsular polysaccharides fromdifferent S. pneumoniae serotypes (suitably conjugated to a carrierprotein). In an embodiment the immunogenic composition comprises 10 to23 capsular polysaccharides from different S. pneumoniae serotypes(suitably conjugated to a carrier protein). In an embodiment, thevaccine may be an 11-valent vaccine. For example, a 11-valent vaccinemay comprise polysaccharides from serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14,18C, 19F and 23F. In an embodiment, the vaccine may be an 12-valent or13-valent vaccine. A 12 or 13-valent paediatric (infant) vaccine mayalso include the 11 valent formulation supplemented with serotypes 19A,or 22F, or 15 (e.g. PS1-PD, PS4-PD, PS5-PD, PS6A-CRM197, PS6B-PD,PS7F-PD, 9V-PD, 14-PD, 18C-TT, 19A-CRM197, 19F-DT, 23F-PD), whereas a13-valent elderly vaccine may include the 11 valent formulationsupplemented with serotypes 19A and 22F, 8 and 12F, or 8 and 15, or 8and 19A, or 8 and 22F, or 12F and 15, or 12F and 19A, or 12F and 22F, or15 and 19A, or 15 and 22F. In an embodiment, the vaccine may be a14-valent or 15-valent vaccine. A 14 or 15-valent paediatric vaccine mayinclude the 11 valent formulation described above supplemented withserotypes 3, 19A and 22F; serotypes 8, 19A and 22F; serotypes 12F, 19Aand 22F; serotypes 15, 19A and 22F; serotypes 3, 8, 19A and 22F;serotypes 3, 12F, 19A and 22F; serotypes 3, 15, 19A and 22F. In anembodiment, the vaccine may be a 16-valent vaccine. A 16 valent vaccinemay include the 11 valent formulation described above supplemented withserotypes 3, 15B, 19A, 22F and 23F. A 16 valent vaccine may include the11 valent formulation described above supplemented with serotypes 3,15B, 19A, 22F and 33F. In an embodiment, the vaccine may be a 19-valentvaccine. A 19 valent vaccine may include the 11 valent formulationdescribed above supplemented with serotypes 8, 10A, 11A, 12F, 15B, 19A,22F and 23F. A 19 valent vaccine may include the 11 valent formulationdescribed above supplemented with serotypes 8, 10A, 11A, 12F, 15B, 19A,22F and 33F. In an embodiment, the vaccine may be a 20-valent vaccine. A20 valent vaccine may include the 11 valent formulation described abovesupplemented with serotypes 3, 8, 10A, 11A, 12F, 15B, 19A, 22F and 23F.A 20 valent vaccine may include the 11 valent formulation describedabove supplemented with serotypes 3, 8, 10A, 11A, 12F, 15B, 19A, 22F and33F. In an embodiment, the vaccine may be a 21-valent vaccine. In anembodiment, the vaccine may be a 22-valent vaccine. In an embodiment,the vaccine may be a 23-valent vaccine.

Suitably, each of the saccharides is conjugated to a carrier protein.Examples of carrier proteins which may be used in the present inventionare TT, DT, CRM197, PhtD, detoxified pneumolysin and protein D. In afurther embodiment, each Streptococcus pneumoniae capsular saccharide isconjugated to a carrier protein independently selected from the groupconsisting of TT, DT, CRM197, PhtD and protein D. In a furtherembodiment, each Streptococcus pneumoniae capsular saccharide isconjugated to a carrier protein independently selected from the groupconsisting of TT, DT, CRM197 and protein D. In an embodiment, theimmunogenic composition of the invention comprises two or more differentcarrier proteins. In an embodiment, the immunogenic composition of theinvention comprises 2, 3, 4, 5 or 6 different carrier proteins.

In an embodiment, the carrier protein is protein D from Haemophilusinfluenzae (PD), for example, protein D sequence from FIG. 9 (FIGS. 9aand 9b together, 364 amino acids) of EP 0594610 (SEQ ID NO: 6).Inclusion of this protein in the immunogenic composition may provide alevel of protection against Haemophilus influenzae related otitis media(Pyrmula et. al. Lancet 367; 740-748 (2006)). The Protein D may be usedas a full length protein or as a fragment (for example, Protein D may beas described in WO0056360). For example, a protein D sequence maycomprise (or consist) of the protein D fragment described in EP0594610which begins at the sequence SSHSSNMANT (SerSerHisSerSerAsnMetAlaAsnThr)(SEQ ID NO. 8), and lacks the 19 N-terminal amino acids from FIG. 9 ofEP0594610, optionally with the tripeptide MDP from NS1 fused to theN-terminal of said protein D fragment (348 amino acids) (SEQ ID NO:7).In one aspect, the protein D or fragment of protein D is unlipidated.The protein D could be present in the immunogenic composition as a freeprotein or as a carrier protein. In one aspect, protein D is present inthe immunogenic composition as free protein. In another aspect, proteinD is present both as a carrier protein and as free protein. In a furtheraspect, protein D is present as a carrier protein for one or more of thepolysaccharides. In a further aspect, 2-9 of the capsularpolysaccharides selected from different serotypes are conjugated toprotein D. In a further aspect, protein D is present as a carrierprotein for the majority of the polysaccharides, for example 6, 7, 8, 9or more of the polysaccharides may be conjugated to protein D.

In an embodiment, the carrier protein is CRM197. CRM197 is a non-toxicform of the diphtheria toxin but is immunologically indistinguishablefrom the diphtheria toxin (DT). Genetically detoxified analogues ofdiphtheria toxin include CRM197 and other mutants described in U.S. Pat.Nos. 4,709,017, 5,843,711, 5,601,827, and 5,917,017. CRM197 is producedby C. diphtheriae infected by the nontoxigenic phase β197tox-created bynitrosoguanidine mutagenesis of the toxigenic carynephage b (Uchida etal Nature New Biology (1971) 233; 8-11). The CRM197 protein has the samemolecular weight as the diphtheria toxin but differs from it by a singlebase change in the structural gene. This leads to a glycine to glutaminechange of amino acid at position 52 which makes fragment A unable tobind NAD and therefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem.46; 69-94, Rappuoli Applied and Environmental Microbiology September1983 p 560-564).

In an embodiment, the carrier protein is Tetanus Toxoid (TT). Tetanustoxin is a single peptide of approximately 150 kDa, which consists of1315 amino-acid residues. Tetanus-toxin may be cleaved by papain toyield two fragments; one of them, fragment C, is approximately 50 kDa.Fragment C of TT is described in Neubauer et al. Biochim. Biophys. Acta1981, 27, 141-148.

Conjugates can be prepared by direct reductive amination methods asdescribed in, US200710184072 (Hausdorff) U.S. Pat. No. 4,365,170(Jennings) and U.S. Pat. No. 4,673,574 (Anderson). Other methods aredescribed in EP-0-161-188, EP-208375 and EP-0-477508. The conjugationmethod may alternatively rely on activation of the saccharide with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. Such conjugates are described in PCT publishedapplication WO 93/15760 Uniformed Services University and WO 95/08348and WO 96/29094. See also Chu C. et al Infect. Immunity, 1983 245 256.The activated saccharide may thus be coupled directly or via a spacer(linker) group to an amino group on the carrier protein. For example,the spacer could be cystamine or cysteamine to give a thiolatedpolysaccharide which could be coupled to the carrier via a thioetherlinkage obtained after reaction with a maleimide-activated carrierprotein (for example using GMBS (4-Maleimidobutyric acidN-hydroxysuccinimide ester)) or a haloacetylated carrier protein (forexample using SIAB (succinimidyl (4-iodoacetyl)aminobenzoate), or SIA(succinimidyl iodoacetate), or SBAP(succinimidyl-3-(bromoacetamide)propionate)). In an embodiment, thecyanate ester (optionally made by CDAP chemistry) is coupled with hexanediamine or ADH (adipic acid dihydrazide) and the amino-derivatisedsaccharide is conjugated to the carrier protein using carbodiimide (e.g.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC or EDC)) chemistryvia a carboxyl group on the protein carrier. Such conjugates aredescribed in PCT published application WO 93/15760 Uniformed ServicesUniversity and WO 95/08348 and WO 96/29094.

In one aspect of the invention, dPly is individually pre-adsorbed ontoaluminium phosphate in accordance with the present invention, before itis mixed with other antigens (for example before mixing withStreptococcus pneumoniae protein PhtD). Thus, in an aspect of theinvention, the process of the invention further comprises the step (iii)mixing the adsorbed detoxified pneumolysin with one or more antigen(s)other than detoxified pneumolysin (e.g. PhtD). In one embodiment, step(iii) is suitably carried out following step (i). In another embodiment,step (iii) is suitably carried out following step (ii). PhtD protein canbe prepared and purified as described in WO2007/071710 (see Example 1b).

In one embodiment, step (iii) comprises mixing the adsorbed detoxifiedpneumolysin with pre-adsorbed PhtD (i.e. PhtD which has previously beenadsorbed onto aluminium phosphate). The PhtD pre-adsorbed onto aluminiumphosphate may have been prepared by a process using different adsorptionconditions from those used for the adsorption of detoxified pneumolysin.Thus, in one aspect, the pre-adsorbed PhtD is preadsorbed onto aluminiumphosphate using different adsorption conditions (e.g. a different pHand/or a different ratio of protein:Al³⁺ (from aluminium phosphate))from the adsorption conditions used for the adsorption of detoxifiedpneumolysin onto aluminium phosphate. For example, in one aspect,pre-adsorbed PhtD is prepared by admixing the PhtD with aluminiumphosphate at pH 4.5 to 5.5, pH 4.5 to 5.4, pH 4.7 to 5.2 or pH 4.9 to5.1 (e.g. pH 5.0) and/or using a ratio of PhtD:Al³⁺ (from aluminiumphosphate) of between 1:1 to 1:3, suitably between 1:1 to 1:2.5, orbetween 1:1.5 to 1:2.5, or between 1:2 to 1:2.5 (e.g. 1:2) (w/w;weight/weight). In another aspect, PhtD is pre-adsorbed onto aluminiumphosphate at pH 4.9 to 5.1 and/or in a ratio of 1 μg of PhtD to 2 μg ofAl³⁺ (from aluminium phosphate). In one aspect, the PhtD is unconjugatedPhtD. In another aspect, the PhtD is conjugated PhtD. Unless otherwisestated, such ranges are inclusive of the end points. In anotherembodiment, pre-adsorbed PhtD is prepared by admixing the PhtD withaluminium phosphate at pH 4.5 to 5.5, pH 4.5 to 5.4, pH 4.7 to 5.2 or pH4.9 to 5.1 and/or using a ratio of PhtD:Al³⁺ (from aluminium phosphate)of between 1:1 to 1:3, suitably between 1:1 to 1:2.5, or between 1:1.5to 1:2.5, or between 1:2 to 1:2.5 (w/w; weight/weight) not including theend points.

In an embodiment, pre-adsorption of PhtD is carried out by a processwherein the PhtD and the aluminium phosphate (and optionally a buffer)are initially mixed and subsequently (e.g. after 5-15 minutes) the pH isadjusted to pH 4.5 to 5.5, pH 4.5 to 5.4, pH 4.7 to 5.2 or pH 4.9 to 5.1(e.g. pH 5.0) (the pH for adsorption), followed by further mixing. ThepH may be adjusted using sodium hydroxide (NaOH (aq)) and hydrochloricacid (HCl (aq)). Suitably, the pH is maintained (and the further mixingcontinues) at this pH (i.e. the pH for adsorption) for between 10minutes to 2 weeks, for example, 10 minutes to 5 hours, 1 to 5 hours, or2 to 3 hours. Unless otherwise stated, such ranges are inclusive of theend points. In another embodiment, the pH is maintained (and the furthermixing continues) at this pH (i.e. the pH for adsorption) for between 10minutes to 2 weeks, for example, 10 minutes to 5 hours, 1 to 5 hours, or2 to 3 hours not including the end points.

In an aspect of the invention, the pre-adsorption of PhtD (i.e. theadmixing of PhtD with aluminium phosphate), is carried out in thepresence of a buffer, such as phosphate buffer (e.g. NaK₂). In oneaspect, the concentration of the buffer (e.g. NaK₂) is at least 1 mM(e.g. at least 1.5 mM, 2 mM, 2.3 mM, 3 mM, 4 mM) and is suitably at most20 mM (e.g. at most 19 mM, 18 mM, 17 mM, 16 mM, 15 mM). In anotheraspect the concentration of the buffer (e.g. NaK₂) is between 1 mM and25 mM, or between 5 mM and 15 mM (e.g. between 8 mM and 12 mM), forexample, 10 mM. The phosphate buffer, NaK₂, used in the adsorption ofPhtD may comprise (sodium phosphate monobasic) NaH₂PO₄.1H₂O and(potassium phosphate dibasic) K₂HPO₄ or K₂HPO₄.3H₂O. Suitably, thebuffer has a pH 6.5 to 7.5 (e.g. pH 7.15). Unless otherwise stated, suchranges are inclusive of the end points. In another embodiment, theconcentration of the buffer (e.g. NaK₂) is between 1 mM and 25 mM, orbetween 5 mM and 15 mM (e.g. between 8 mM and 12 mM) not including theend points.

In an aspect of the invention, following pre-adsorption of PhtD ontoaluminium phosphate, the pH of the pre-adsorbed PhtD is adjusted to a pHbetween 6 and 7 (for example pH 5.9 to 6.5, pH 5.9 to 6.3, pH 6.0) priorto mixing the pre-adsorbed PhtD and pre-adsorbed dPly.

In another aspect of the invention, a mixture of pre-adsorbed dPly andPhtD, may be prepared, according to steps (i) to (iii) described above,prior to mixing with further antigens, e.g. S. pneumoniae capsularsaccharides (suitably conjugated to a carrier protein) as describedherein.

Dosage

The total content of protein antigens in the immunogenic composition orvaccine of the invention will typically be in the range 1-100 μg, or5-80 μg, e.g. in the range 50-70 μg. In one aspect, the immunogeniccomposition or vaccine of the invention comprises 1 μg-50 μg (forexample 26 μg-45 μg, 26 μg-40 μg, 28 μg-35 μg or around 30 μg) ofdetoxified pneumolysin (e.g. dPly), per human dose. In another aspect,the immunogenic composition or vaccine of the invention comprises 1μg-50 μg (for example 26 μg-45 μg, 26 μg-40 μg, 28 μg-35 μg or around 30μg) of each S. pneumoniae protein, per human dose. For example, theimmunogenic composition or vaccine of the invention may comprise 1 μg-50μg (for example 26 μg-45 μg, 26 μg-40 μg, 28 μg-35 μg or around 30 μg)of PhtD, per human dose.

In an embodiment, the immunogenic composition or vaccine of theinvention may comprise S. pneumoniae capsular saccharides, each of whichmay be at a dose of between 0.1-20 μg; 0.5-10 μg; 0.5-5 μg or 1-3 μg ofsaccharide. In an embodiment, capsular polysaccharides may be present atdifferent dosages, for example some capsular polysaccharides may bepresent at a dose of around or exactly 1 μg or some capsularpolysaccharides may be present at a dose of around or exactly 3 μg.“Around” or “approximately” are defined as within 10% more or less ofthe given figure for the purposes of the invention.

By the term “human dose” is meant a dose which is in a volume suitablefor human use. Generally this is between 0.25 and 1.5 ml, although, foradministration to the skin a lower volume of between 0.05 ml and 0.2 mlmay be used. In one embodiment, a human dose is 0.5 ml. In a furtherembodiment, a human dose is higher than 0.5 ml, for example 0.6, 0.7,0.8, 0.9 or 1 ml. In a further embodiment, a human dose is between 1 mland 1.5 ml. In another embodiment, in particular when the immunogeniccomposition is for the paediatric population, a human dose may be lessthan 0.5 ml such as between 0.25 and 0.5 ml.

Method of Administration

The vaccine preparations containing immunogenic compositions of thepresent invention may be used to protect or treat a mammal, e.g. human,susceptible to infection, by means of administering said vaccine via asystemic or mucosal route. These administrations may include injectionvia the intramuscular (IM), intraperitoneal (IP), intradermal (ID) orsubcutaneous (SC) routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts. Although the vaccineof the invention may be administered as a single dose, componentsthereof may also be co-administered together at the same time or atdifferent times (for instance pneumococcal saccharide conjugates couldbe administered separately, at the same time or 1-2 weeks after theadministration of the any bacterial protein component of the vaccine foroptimal coordination of the immune responses with respect to eachother). For co-administration, the optional adjuvant may be present inany or all of the different administrations. In addition to a singleroute of administration, 2 different routes of administration may beused. For example, polysaccharide conjugates may be administered IM (orID) and bacterial proteins may be administered IN (or ID). In addition,the vaccines of the invention may be administered IM for priming dosesand IN for booster doses.

Following an initial vaccination, subjects may receive one or severalbooster immunizations adequately spaced.

Vaccine

The present invention further provides a vaccine containing theimmunogenic compositions of the invention and a pharmaceuticallyacceptable excipient or carrier.

Pharmaceutically acceptable excipients and carriers are well known andcan be selected by those of skill in the art. For example, thepharmaceutically acceptable excipient or carrier can include a buffer,such as Tris (trimethamine), phosphate (e.g. sodium phosphate), acetate,borate (e.g. sodium borate), citrate, glycine, histidine and succinate(e.g. sodium succinate), suitably sodium chloride, histidine, sodiumphosphate or sodium succinate. The pharmaceutically acceptable excipientmay include a salt, for example sodium chloride, potassium chloride ormagnesium chloride. Optionally, the pharmaceutically acceptableexcipient contains at least one component that stabilizes solubilityand/or stability. Examples of solubilizing/stabilizing agents includedetergents, for example, laurel sarcosine and/or polysorbate (e.g.Tween™ 80). Examples of stabilizing agents also include poloxamer (e.g.poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer407). The phamaceutically acceptable excipient may include a non-ionicsurfactant, for example polyoxyethylene sorbitan fatty acid esters,Polysorbate-80 (Tween™ 80), Polysorbate-60 (Tween™ 60), Polysorbate-40(Tween™ 40) and Polysorbate-20 (Tween™ 20), or polyoxyethylene alkylethers (suitably polysorbate-80). Alternative solubilizing/stabilizingagents include arginine, and glass forming polyols (such as sucrose,trehalose and the like). The pharmaceutically excipient may be apreservative, for example phenol, 2-phenoxyethanol, or thiomersal. Otherpharmaceutically acceptable excipients include sugars (e.g. lactose,sucrose), and proteins (e.g. gelatine and albumin). Pharmaceuticallyacceptable carriers include water, saline solutions, aqueous dextroseand glycerol solutions. Numerous pharmaceutically acceptable excipientsand carriers are known in the art and are described, e.g., inRemington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 5th Edition (975).

According to a further aspect of the invention there is provided aprocess for making the immunogenic composition or vaccine of theinvention comprising the step of mixing detoxified pneumolysin adsorbedonto aluminium phosphate according to the invention with apharmaceutically acceptable excipient or carrier.

The vaccines of the present invention may be stored in solution orlyophilized. In an embodiment, the solution is lyophilized in thepresence of a sugar such as sucrose or lactose. It is still furtherpreferable that they are lyophilized and extemporaneously reconstitutedprior to use. Lyophilizing may result in a more stable composition(vaccine) and may possibly lead to higher antibody titers in thepresence of 3D-MPL and in the absence of an aluminum based adjuvant.

The vaccine or immunogenic composition of the invention may alsocomprise an antimicrobial, typically when package in multiple doseformat. For example, the immunogenic composition or vaccine of theinvention may comprise 2-phenoxyethanol.

The vaccine or immunogenic composition of the invention may alsocomprise a detergent e.g. polysorbate, such as Tween™ 80. Detergents aregenerally present at low levels e.g. <0.01%, but higher levels have beensuggested for stabilising antigen formulations e.g. up to 10%.

In one aspect of the invention is provided a vaccine kit, comprising avial containing an immunogenic composition of the invention, optionallyin lyophilised form, and further comprising a vial containing anadjuvant as described herein. It is envisioned that in this aspect ofthe invention, the adjuvant will be used to reconstitute the lyophilisedimmunogenic composition.

Although the vaccines of the present invention may be administered byany route, administration of the described vaccines into the skin (ID)forms one embodiment of the present invention. Human skin comprises anouter “horny” cuticle, called the stratum corneum, which overlays theepidermis. Underneath this epidermis is a layer called the dermis, whichin turn overlays the subcutaneous tissue. Researchers have shown thatinjection of a vaccine into the skin, and in particular the dermis,stimulates an immune response, which may also be associated with anumber of additional advantages. Intradermal vaccination with thevaccines described herein forms a preferred feature of the presentinvention.

The conventional technique of intradermal injection, the “mantouxprocedure”, comprises steps of cleaning the skin, and then stretchingwith one hand, and with the bevel of a narrow gauge needle (26-31 gauge)facing upwards the needle is inserted at an angle of between 10-15°.Once the bevel of the needle is inserted, the barrel of the needle islowered and further advanced whilst providing a slight pressure toelevate it under the skin. The liquid is then injected very slowlythereby forming a bleb or bump on the skin surface, followed by slowwithdrawal of the needle.

More recently, devices that are specifically designed to administerliquid agents into or across the skin have been described, for examplethe devices described in WO 99/34850 and EP 1092444, also the jetinjection devices described for example in WO 01/13977; U.S. Pat. Nos.5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189,5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335,5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941,880,4,940,460, WO 97/37705 and WO 97/13537. Alternative methods ofintradermal administration of the vaccine preparations may includeconventional syringes and needles, or devices designed for ballisticdelivery of solid vaccines (WO 99/27961), or transdermal patches (WO97/48440; WO 98/28037); or applied to the surface of the skin(transdermal or transcutaneous delivery WO 98/20734; WO 98/28037).

When the vaccines of the present invention are to be administered to theskin, or more specifically into the dermis, the vaccine is in a lowliquid volume, particularly a volume of between about 0.05 ml and 0.2ml.

The content of the immunogenic composition in the skin or intradermalvaccines of the present invention may be similar to conventional dosesas found in intramuscular vaccines (see above). However, it is a featureof skin or intradermal vaccines that the formulations may be “low dose”.Accordingly the protein antigens in “low dose” vaccines are suitablypresent in as little as 0.1 to 10 μg, or 0.1 to 5 μg per dose; and thepolysaccharide (suitably conjugated) antigens may be present in therange of 0.01-1 μg, and suitably between 0.01 to 0.5 μg of saccharideper dose.

As used herein, the term “intradermal delivery” means delivery of thevaccine or immunogenic composition to the region of the dermis in theskin. However, the vaccine or immunogenic composition will notnecessarily be located exclusively in the dermis. The dermis is thelayer in the skin located between about 1.0 and about 2.0 mm from thesurface in human skin, but there is a certain amount of variationbetween individuals and in different parts of the body. In general, itcan be expected to reach the dermis by going 1.5 mm below the surface ofthe skin. The dermis is located between the stratum corneum and theepidermis at the surface and the subcutaneous layer below. Depending onthe mode of delivery, the vaccine or immunogenic composition mayultimately be located solely or primarily within the dermis, or it mayultimately be distributed within the epidermis and the dermis.

The present invention further provides an improved vaccine for theprevention or amelioration of otitis media caused by Haemophilusinfluenzae by the addition of Haemophilus influenzae proteins, forexample protein D in conjugated form or as a free (unconjugated)protein. One or more Moraxella catarrhalis protein antigens can also beincluded in the vaccine or immunogenic composition of the invention in afree or conjugated form. Thus, the present invention is an improvedmethod to elicit an immune response against otitis media in infants.

Examples of preferred Moraxella catarrhalis protein antigens which canbe included in a combination vaccine or immunogenic composition of theinvention (especially for the prevention of otitis media) are: outermembrane protein 106 (OMP106) [WO 97/41731 (Antex) & WO 96/34960 (PMC)];outer membrane protein 21 (OMP21) or fragments thereof (WO 0018910);lactoferrin binding protein A (LbpA) &/or lactoferrin binding protein B(LbpB) [WO 98/55606 (PMC)]; transferrin binding protein A (TbpA) &/ortransferring binding protein B (TbpB) [WO 97/13785 & WO 97/32980 (PMC)];Moraxella catarrhalis CopB protein [Helminen M E, et al. (1993) Infect.Immun. 61:2003-2010]; ubiquitous surface protein A1 (UspA1) and/orubiquitous surface protein A2 (UspA2) [WO 93/03761 (University ofTexas)]; outer membrane protein CD (OmpCD); HasR (PCT/EP99/03824); PilQ(PCT/EP99/03823); outer membrane protein 85 (OMP85) (PCT/EP00/01468);lipo06 (GB 9917977.2); lipo10 (GB 9918208.1); lipo11 (GB 9918302.2);lipo18 (GB 9918038.2); outer membrane protein P6 (P6) (PCT/EP99/03038);D15 surface antigen (D15) (PCT/EP99/03822); outer membrane protein A1(OmpA1) (PCT/EP99/06781); Hly3 (PCT/EP99/03257); and outer membraneprotein E (OmpE). Examples of non-typeable Haemophilus influenzaeproteins or fragments thereof which can be included in a combinationvaccine (especially for the prevention of otitis media) include: Fimbrinprotein [(U.S. Pat. No. 5,766,608—Ohio State Research Foundation)] andfusions comprising peptides therefrom [eg LB1(f) peptide fusions; U.S.Pat. No. 5,843,464 (OSU) or WO 99/64067]; outer membrane protein 26(OMP26) [WO 97/01638 (Cortecs)]; P6 [EP 281673 (State University of NewYork)]; TbpA and/or TbpB; H, influenzae adhesin (Hia); Haemophilussurface fibrils (Hsf); Haemophilus influenza Hin47 protein; Haemophilusinfluenzae Hif protein; Haemophilus influenzae Hmw1 protein; Haemophilusinfluenzae Hmw2 protein; Haemophilus influenzae Hmw3 protein;Haemophilus influenzae Hmw4 protein; Haemophilus influenzaeautotransporter adhesin (Hap); D15 (WO 94/12641); P2; and P5 (WO94/26304).

Methods of Treatment and Use

The present invention provides a method for the treatment or preventionof Streptococcus pneumoniae infection in a subject in need thereofcomprising administering to said subject a therapeutically effectiveamount of an immunogenic composition or the vaccine of the invention.The present invention also provides a method of immunising a human hostagainst Streptococcus pneumoniae infection comprising administering tothe host an immunoprotective dose of the immunogenic composition orvaccine of the invention. The present invention also provides a methodof inducing an immune response to Streptococcus pneumoniae (e.g.Streptococcus pneumoniae pneumolysin) in a subject, the methodcomprising administering a therapeutically effective amount of theimmunogenic composition or vaccine of the invention.

In an embodiment, the present invention is an improved method to elicitan immune response in infants (defined as 0-2 years old in the contextof the present invention) by administering a therapeutically effectiveamount of an immunogenic composition or vaccine of the invention (apaediatric vaccine). In one embodiment, the vaccine is a paediatricvaccine. In one embodiment, the immune response is protective (i.e. itcan prevent or reduce infection caused by S. pneumoniae).

In an embodiment, the present invention is an improved method to elicitan immune response in the elderly population (in the context of thepresent invention a patient is considered elderly if they are 50 yearsor over in age, typically over 55 years and more generally over 60years) by administering a therapeutically effective amount of theimmunogenic composition or vaccine of the invention.

In one embodiment, the present invention provides a method of protectinga subject against a disease caused by infection with Streptococcuspneumoniae, or a method of preventing infection with Streptococcuspneumoniae, or a method of reducing the severity of or delaying theonset of at least one symptom associated with an infection caused byStreptococcus pneumoniae, the methods comprising administering to asubject an immunogenic amount of an immunogenic composition or vaccineof the invention.

In an embodiment, the present invention provides immunogeniccompositions and vaccines of the invention for use in the prevention ortreatment of a disease caused by S. pneumoniae infection. In anembodiment, the present invention provides the use of an immunogeniccomposition or vaccine of the invention in the manufacture of amedicament for the prevention (or treatment) of a disease caused by S.pneumoniae infection.

The disease caused by Streptococcus pneumoniae infection may be selectedfrom pneumonia, invasive pneumococcal disease (IPD), exacerbations ofchronic obstructive pulmonary disease (eCOPD), otitis media, meningitis,bacteraemia, pneumonia and/or conjunctivitis. Where the human host is aninfant (defined as 0-2 years old in the context of the presentinvention), the disease may be selected from otitis media, meningitis,bacteraemia, pneumonia and/or conjunctivitis. In one aspect, where thehuman host is an infant (defined as 0-2 years old in the context of thepresent invention), the disease is selected from otitis media and/orpneumonia. Where the human host is elderly (i.e. 50 years or over inage, typically over 55 years and more generally over 60 years), thedisease may be selected from pneumonia, invasive pneumococcal disease(IPD), and/or exacerbations of chronic obstructive pulmonary disease(eCOPD). In one aspect, where the human host is elderly, the disease isinvasive pneumococcal disease (IPD). In another aspect, where the humanhost is elderly, the disease is exacerbations of chronic obstructivepulmonary disease (eCOPD).

Embodiments herein relating to “vaccine compositions” of the inventionare also applicable to embodiments relating to “immunogeniccompositions” of the invention, and vice versa.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

Embodiments of the invention are further described in the subsequentnumbered paragraphs:

-   -   1. An immunogenic composition comprising detoxified pneumolysin        adsorbed onto aluminium phosphate, wherein more than 85% (e.g.        more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of        the detoxified pneumolysin is adsorbed onto aluminium phosphate.    -   2. An immunogenic composition according to paragraph 1, wherein        more than 95% of the detoxified pneumolysin is adsorbed onto        aluminium phosphate    -   3. An immunogenic composition according to paragraph 1 or        paragraph 2, wherein the immunogenic composition has a pH        between 6 and 7 (e.g. pH 6.0 to 6.5, pH 6.0 to 6.2, pH 6.1).    -   4. An immunogenic composition according to any one of paragraphs        1 to 3, wherein greater than 80% (e.g. more than 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89% or 90%) of the detoxified        pneumolysin adsorbed onto aluminium phosphate has a particle        size less than 10 μm.    -   5. An immunogenic composition according to any one of paragraphs        1 to 4, wherein greater than 85% of the detoxified pneumolysin        adsorbed onto aluminium phosphate has a particle size less than        10 μm.    -   6. An immunogenic composition according to any one of paragraphs        1 to 5, wherein the detoxified pneumolysin has been chemically        detoxified.    -   7. An immunogenic composition according to any one of paragraphs        1 to 6, wherein the detoxified pneumolysin has been genetically        detoxified.    -   8. An immunogenic composition according to any one of paragraphs        1 to 7, wherein the detoxified pneumolysin is unconjugated.    -   9. An immunogenic composition according to any one of paragraphs        1 to 8, wherein the detoxified pneumolysin is conjugated to a        saccharide, for example a capsular saccharide of S. pneumoniae.    -   10. An immunogenic composition according to any one of        paragraphs 1 to 9, further comprising PhtD adsorbed onto        aluminium phosphate.    -   11. An immunogenic composition according to paragraph 10,        wherein the PhtD is unconjugated.    -   12. An immunogenic composition according to paragraph 10,        wherein the PhtD is conjugated to a saccharide, for example a        capsular saccharide of S. pneumoniae.    -   13. An immunogenic composition according to any one of        paragraphs 1 to 12 further comprising 10 or more S. pneumoniae        capsular polysaccharides from different S. pneumoniae serotypes        conjugated to carrier protein(s).    -   14. A process for adsorption of detoxified pneumolysin onto        aluminium phosphate comprising the step of (i) admixing        detoxified pneumolysin and the aluminium phosphate at a pH less        than 6.5 (for example, less than pH 6.0, pH 5.0 to 6.2, pH 5.0        to 6.1, pH 5.2 to 6.2, pH 5.2 to 6.1, pH 5.4 to 6.2, pH 5.4 to        6.1, pH 5.5 to 6.1, pH 5.4 to 5.9, pH 5.5 to 5.9, pH 5.4 to 5.7,        pH 5.5 to 5.7, pH 5.4 to 5.6 or pH 5.5).    -   15. The process according to paragraph 14 wherein the detoxified        pneumolysin and aluminium phosphate are in a ratio of dPly:Al³⁺        (from aluminium phosphate) in step (i) between 1:1.5 to 1:4        (e.g. 1:1.5 to 1:3.5, 1:1.5 to 1:2.5, 1:2 to 1:2.5, 1:2.5 to        1:3.5, 1:3 to 1:3.5, 1:2 or 1:3) (w/w; weight/weight).    -   16. The process according to any of paragraphs 14 or 15 wherein        step (i) is carried out in the presence of a phosphate buffer,        optionally comprising NaH₂ PO₄ and K₂HPO₄, and optionally at a        concentration of between 1 mM and 5 mM (e.g. between 1 mM and 3        mM, between 2 mM and 2.4 mM, or 2 mM).    -   17. The process according to any one of paragraphs 14 to 16        followed by step (ii) adjustment of the pH of the composition to        a pH between 6 and 7 (e.g. pH 6.0 to 6.5, pH 6.0 to 6.3, or pH        6.1).    -   18. The process according to any one of paragraphs 14 to 17        followed by step (iii) mixing the adsorbed detoxified        pneumolysin with one or more antigen(s) other than detoxified        pneumolysin (e.g. PhtD).    -   19. The process according to paragraph 18 wherein step (iii)        comprises mixing the adsorbed detoxified pneumolysin with        pre-adsorbed PhtD.    -   20. The process according to paragraph 19 wherein pre-adsorbed        PhtD is prepared by admixing PhtD with aluminium phosphate at pH        4.5 to 5.5 (e.g. pH 4.5 to 5.4, pH 4.7 to 5.2, pH 4.9 to 5.1, or        pH 5.0) and/or using a ratio of PhtD:Al³⁺ (from aluminium        phosphate) of between 1:1 to 1:3 (e.g. 1:1 to 1:2.5, 1:1.5 to        1:2.5, 1:2 to 1:2.5, or 1:2) (w/w; weight/weight).    -   21. The process according to paragraph 20 wherein admixing PhtD        with aluminium phosphate is carried out in the presence of a        phosphate buffer, optionally comprising NaH₂PO₄.1H₂O, K₂HPO₄        and/or K₂HPO₄.3H₂O, and optionally at a concentration between 5        mM and 15 mM (e.g. between 8 mM and 12 mM, or 10 mM).    -   22. The process according to any one of paragraphs 19 to 21        wherein the PhtD is conjugated to a saccharide, for example a        capsular saccharide of S. pneumoniae.    -   23. The process according to any one of paragraphs 19 to 21        wherein the PhtD is unconjugated.    -   24. The process according to any one of paragraphs 14 to 23        wherein the detoxified pneumolysin is unconjugated.    -   25. The process according to any one of paragraphs 14 to 23        wherein the detoxified pneumolysin is conjugated to a        saccharide, for example a capsular saccharide of S. pneumoniae.    -   26. A process for preparing an immunogenic composition        comprising detoxified pneumolysin, comprising the process of        paragraphs 14 to 25.    -   27. An immunogenic composition according to any one of        paragraphs 1 to 13 prepared by the process according to        paragraphs 14 to 26.    -   28. A vaccine comprising the immunogenic composition of any one        of paragraphs 1 to 13 or 27 and a pharmaceutically acceptable        excipient or carrier.    -   29. A method for the treatment or prevention of Streptococcus        pneumoniae infection in a subject in need thereof (e.g. human)        comprising administering to said subject a therapeutically        effective amount of an immunogenic composition of any of        paragraphs 1 to 13 or 27 or the vaccine of paragraph 28.    -   30. A method of immunising a human host against Streptococcus        pneumoniae infection comprising administering to the host an        immunoprotective dose of the immunogenic composition of any of        paragraphs 1 to 13 or 27 or vaccine of paragraph 28.    -   31. A method of inducing an immune response to Streptococcus        pneumoniae in a subject (e.g. human), the method comprising        administering a therapeutically or prophylactically effective        amount of the immunogenic composition of any of paragraphs 1 to        13 or 27 or the vaccine of paragraph 28.    -   32. The immunogenic composition of paragraphs 1 to 13 or 27 or        vaccine of paragraph 28 for use in the treatment or prevention        of disease caused by Streptococcus pneumoniae infection.    -   33. A use of the immunogenic composition of paragraphs 1 to 13        or 27 or vaccine of paragraph 28 in the manufacture of a        medicament for the treatment or prevention of a disease caused        by Streptococcus pneumoniae infection.

Further embodiments of the invention are also described in thesubsequent numbered paragraphs:

-   -   1a. An immunogenic composition comprising detoxified pneumolysin        adsorbed onto aluminium phosphate, wherein more than 85% (e.g.        more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) of        the detoxified pneumolysin is adsorbed onto aluminium phosphate.    -   2a. An immunogenic composition according to paragraph 1a,        wherein the pH of the composition is between 6 and 7 (e.g. pH        6.0 to 6.5, pH 6.0 to 6.2, pH 6.1).    -   3a. An immunogenic composition according to paragraph 1a or        paragraph 2a, wherein greater than 80% (e.g. more than 81%, 82%,        83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%) of the particles of        detoxified pneumolysin adsorbed onto aluminium phosphate have a        size less than 10 μm.    -   4a. An immunogenic composition according to any one of        paragraphs 1a to 3a, wherein the pneumolysin has been chemically        detoxified.    -   5a. An immunogenic composition according to any one of        paragraphs 1a to 4a, wherein the pneumolysin has been        genetically detoxified.    -   6a. A process for adsorption of detoxified pneumolysin onto        aluminium phosphate comprising the step of (i) admixing        detoxified pneumolysin and the aluminium phosphate at a pH less        than 6.5 (for example, less than pH 6.0, pH 5.0 to 6.2, pH 5.0        to 6.1, pH 5.2 to 6.2, pH 5.2 to 6.1, pH 5.4 to 6.2, pH 5.4 to        6.1, pH 5.5 to 6.1, pH 5.4 to 5.9, pH 5.5 to 5.9, pH 5.4 to 5.7,        pH 5.5 to 5.7, pH 5.4 to 5.6 or pH 5.5).    -   7a. The process according to paragraph 6a wherein the ratio of        dPly:Al³⁺ (from aluminium phosphate) in step (i) is between        1:1.5 to 1:4 (e.g. 1:1.5 to 1:3.5, 1:1.5 to 1:2.5, 1:2 to 1:2.5,        1:2.5 to 1:3.5, 1:3 to 1:3.5, 1:2 or 1:3) (w/w; weight/weight).    -   8a. The process according to paragraph 6a or 7a step (i) is        carried out in the presence of a phosphate buffer, optionally        comprising NaH₂PO₄ and K₂HPO₄, and optionally at a concentration        of between 1 mM and 5 mM (e.g. between 1 mM and 3 mM, between 2        mM and 2.4 mM, or 2 mM).    -   9a. The process according to any one of paragraphs 6a to 8a        followed by step (ii) adjustment of the pH of the composition to        a pH between 6 and 7 (e.g. pH 6.0 to 6.5, pH 6.0 to 6.3, or pH        6.1).    -   10a. The process according to any one of paragraphs 6a to 9a        followed by step (iii) mixing the adsorbed detoxified        pneumolysin with one or more antigen(s) other than detoxified        pneumolysin (e.g. PhtD).    -   11a. The process according to paragraph 10a wherein step (iii)        comprises mixing the adsorbed detoxified pneumolysin with        pre-adsorbed PhtD.    -   12a. The process according to paragraph 11a wherein pre-adsorbed        PhtD is prepared by admixing PhtD with aluminium phosphate at pH        4.5 to 5.5 (e.g. pH 4.5 to 5.4, pH 4.7 to 5.2, pH 4.9 to 5.1, or        pH 5.0) and/or using a ratio of PhtD:Al³⁺ (from aluminium        phosphate) of between 1:1 to 1:3 (e.g. 1:1 to 1:2.5, 1:1.5 to        1:2.5, 1:2 to 1:2.5, or 1:2) (w/w; weight/weight).    -   13a. The process according to paragraph 12a wherein admixing        PhtD with aluminium phosphate is carried out in the presence of        a phosphate buffer, optionally comprising NaH₂PO_(4.1)H₂O,        K₂HPO₄ and/or K₂HPO₄.3H₂O, and optionally at a concentration        between 5 mM and 15 mM (e.g. between 8 mM and 12 mM, or 10 mM).    -   14a. A process for preparing an immunogenic composition        comprising detoxified pneumolysin, comprising the process of        paragraphs 6a to 13a.    -   15a. An immunogenic composition according to any one of        paragraphs 1a to 5a prepared by the process according to        paragraphs 6a to 14a.    -   16a. A vaccine comprising the immunogenic composition of any one        of paragraphs 1a to 5a or 15a and a pharmaceutically acceptable        excipient or carrier.    -   17a. A method for the treatment or prevention of Streptococcus        pneumoniae infection in a subject in need thereof comprising        administering to said subject a therapeutically effective amount        of an immunogenic composition of any of paragraphs 1a to 5a or        15a or the vaccine of paragraph 16a.    -   18a. A method of immunising a human host against Streptococcus        pneumoniae infection comprising administering to the host an        immunoprotective dose of the immunogenic composition of any of        paragraphs 1a to 5a or 15a or vaccine of paragraph 16a.    -   19a. A method of inducing an immune response to Streptococcus        pneumoniae in a subject, the method comprising administering a        therapeutically or prophylactically effective amount of the        immunogenic composition of any of paragraphs 1a to 5a or 15a or        the vaccine of paragraph 16a.    -   20a. The immunogenic composition of paragraphs 1a to 5a or 15a        or vaccine of paragraph 16a for use in the treatment or        prevention of disease caused by Streptococcus pneumoniae        infection.    -   21a. A use of the immunogenic composition of paragraphs 1a to 5a        or 15a or vaccine of paragraph 16a in the manufacture of a        medicament for the treatment or prevention of a disease caused        by Streptococcus pneumoniae infection.

EXAMPLES Example 1a: Adsorption of Detoxified Pneumolysin onto AluminiumPhosphate

Detoxification of pneumolysin using formaldehyde: A stock of purifiedpneumolysin at a concentration of approximately 0.4 mg/mi was in 25 mMpotassium phosphate buffer pH 7.0 was treated with 50 mM L-lysine and0.1% formaldehyde (w/v) for 21 days at 40° C.

Aluminium phosphate (AlPO₄), 1890 μg Al³⁺/ml together with PO₄ (Na/K₂) 2mM pH7.15 and dPly (detoxified pneumolysin) 630 μg dPly/ml (ratio 1 μgdPly/3 μg Al³⁺) were mixed under magnetic stirring (130 rpm) for 5 to 15minutes at room temperature (18-24° C.). The pH was adjusted to pH5.5+/−0.1 with NaOH 0.05M or 0.5M/HCl 0.03M or 0.3M with magneticstirring (130 rpm) for 5 to 15 minutes at room temperature (18-24° C.).The pH was maintained at pH 5.5+/−0.1 for 120-150 minutes at roomtemperature (18-24° C.) under magnetic stirring (130 rpm). The pH wasthen adjusted to pH 6.1+/−0.1 with NaOH 0.05M or 0.5M/HCl 0.03M or 0.3Mwith magnetic stirring (130 rpm) for 5 to 15 minutes at room temperature(18-24° C.). Maturation was carried out for at least 7 days at 2-8° C.(maturation step) with no agitation.

Method:

AlPO₄ (AP)  →1890 μg Al³⁺/m1 + PO₄ (Na/K₂) 2 mM pH 7.15 + dPly     →630μg dPly/ml (ratio 1 μg dPly/3 μg Al³⁺) ↓(stirring Magnetic—Time (min):5-15—Temp (° C.): Room temp 18-24° C.) Adjust pH 5.5 +/− 0.1 with NaOH0.05M or 0.5M/HCl 0.03M or 0.3M ↓(stirring Magnetic—Time (min):5-15—Temp (° C.): Room temp 18-24° C.) Check pH and adjust if necessary↓(stirring Magnetic—Time (min): 120-150—Temp (° C.): Room temp 18-24°C.) Adjust pH 6.1 +/− 0.1 with NaOH 0.05M or 0.5M/HCl 0.03M or 0.3M↓(stirring Magnetic—Time (min): 5-15—Temp (° C.): Room temp 18-24° C.)Check pH and adjust if necessary ↓ Maturation: Time (min): for at least7 days—Temp (° C.): +2 to +8—Agitation: no ↓ Sampling Remark: thevaccine bulk is maintained under gentle stirring during all formulationprocess, room temperature is 18-24° C.

TABLE 1 Ingredients Name Component Concentration Other Antigen dPly 630μg/ml Al³⁺ from AlPO₄ 1890 μg/ml NaCl 55 mM PO₄ Na/K₂ NaH₂PO₄ 2.4 mMK₂HPO₄ pH 6.1 (+/− 0.1)

Specifications: aluminium 0.50%, phosphate 1.59%, NaCl 0.9%.

Example 1 b: Adsorption of PhtD onto Aluminium Phosphate

The PhtD was taken from storage at −70° C. and thawed in athermostatized bath at 25° C. Aluminium phosphate (AlPO₄), 4000 μgAl³⁺/ml together with PO₄ (Na/K₂) 10 mM pH 7.15 and PhtD 2000 μg PhtD/ml(ratio PhtD/Al³⁺ 1:2) were mixed under magnetic stirring (130 rpm) for 5to 15 minutes at room temperature (18-24° C.). The pH was adjusted to pH5.0+/−0.1 with HCl 0.03M or 0.3M followed by magnetic stirring (130 rpm)for 120-150 minutes at room temperature (18-24° C.). The pH was thenadjusted to pH 6.0+/−0.1 with NaOH 0.05M or 0.5M. Sampling was carriedout followed by maturation for at least 7 days at 2-8° C. (maturationstep) with no agitation.

Method:

Non pH adjusted AlPO₄ (A³⁺)  →4000 μg Al³⁺/m1 + PO₄ (Na/K₂) 10 mM pH7.15 + PhtD           →2000 μg PhtD/ml (ratio PhtD/Al³⁺ 1:2) ↓(stirringMagnetic—Time (min): 5-15—Temp (° C.): Room temp) Adjust and check pH5.0 +/− 0.1 with HCl 0.03M or 0.3M ↓(stirring Magnetic—Time (min):120-150—Temp (° C.): Room temp) Adjust and check pH 6.0 +/− 0.1 withNaOH 0.05M or 0.5M ↓ Sampling ↓ Maturation: Time (min): for at least 7days—Temp (° C.): +2 to +8—Agitation: no The frozen bulk of PhtD(storage temperature: −70° C.) is thawed in a thermostatized bath at 25°C. Remark: the vaccine bulk is maintained under stirring during allformulation process, room temperature is 18-24° C.

TABLE 2 Ingredients Name Component Concentration Other Antigen PhtD 2000μg/ml Al³⁺ AlPO₄ 4000 μg Al³⁺/ml NaCl Residual (+/− 120 mM) PO₄ Na/K₂NaH₂PO₄•2H₂O Residual (+/− K₂HPO₄ or 4.56 mM) K₂HPO₄•3H₂O pH 6.0 (+/−0.1)

DPly/PhtD-AlPO₄ vaccine may be formulated by mixing sterile solutions ofNaCl and water for injection (to reach a final concentration of 150 mMNaCl), prior to addition of the required amount of sterile AlPO₄ whichis added to obtain a final concentration of 0.5 mg Al³⁺ per unit dose(0.5 ml) of final vaccine. The mixture is stirred, the pH is adjusted to6.1±0.1 and the adsorbed dPly and PhtD are added. After addition of theantigens monobulks, the mixture is gently stirred at room temperatureand if needed, the pH is adjusted to 6.1±0.1 before storage of the finalbulk in glass containers at 2-8° C.

Example 1c: Formulation of dPly and PhtD Adsorbed onto AluminiumPhosphate

Antigen was pre-adsorbed separately on AlPO₄ and then pooled to get thefinal vaccine according to the following method:

Method:

WFI (water for injection) + NaCl 1500 mM    →ad 150 mM + AlPO₄  →ad 1000μg Al³⁺/m1 ↓(stirring Magnetic—Time (min): 5-15—Temp (° C.): Room temp)Adjust and check pH 6.1 +/− 0.1 with NaOH 0.05M or 0.5M/HCl 0.03M or0.3M ↓(stirring Magnetic—Time (min): 5-15—Temp (° C.): Room temp)Pre-adsorbed PhtD (on AlPO₄)     →60 μg PhtD/ml Pre-adsorbed dPly (onAlPO₄)    →60 μg dPly/ml ↓(stirring Magnetic—Time (min):15-20—Temp (°C.): Room temp) Check or adjust pH 6.1 +/− 0.1 with NaOH 0.05M or0.5M/HCl 0.03M or 0.3M ↓ Sampling ↓ Storage at Temp (° C.): +2 to +8Remark: the vaccine bulk is maintained under stirring during allformulation process. Note: ad means “up to”.

TABLE 3 Ingredients Name Component Concentration Other Antigen PhtD 60μg/ml of 30 μg of each dPly each protein protein per human dose bothproteins are 300 μg Al³⁺/m1 150 μg Al³⁺ pre-adsorbed per human on AlPO₄dose Al³⁺ AlPO₄  1000 μg/ml Total: 500 μg Al³⁺ per human dose NaCl 150mM 4.38 mg per human dose PO₄ Na/K₂ NaH₂PO₄ Residual K₂HPO₄ Water for ad500 μl injection pH 6.1 (+/− 0.1)

Example 2: Completeness of Adsorption Method:

Completeness of adsorption was measured by measuring the supernatant(SN) of centrifuged samples via Lowry. To assess the percentage of eachantigen adsorbed to the adjuvant (aluminium phosphate), formulationsamples (with dPly) of 250 μl were centrifuged for about 10 minutes at6000 rpm to separate the unadsorbed protein (pellet) from the adsorbedprotein (supernatant). 210 μl of the supernatant (SN1) was collected forthe determination of completeness of adsorption. The proteinconcentration in the supernatant was determined by Lowry (see details ofmethod below). The percentage of adsorption was determined by comparingthe amount of detoxified pneumolysin protein in the supernatant aftercentrifugation compared to a control (unadsorbed detoxifiedpneumolysin). The percentage of adsorption was calculated as follows: %A=100−([PrSN]×100/[PfCtr]) where, [PrSN] is the concentration of proteinin supernatant and [PfCtr] is the concentration in the correspondingunadjuvanted control.

Completeness of adsorption was measured the day of formulation (T0),after 21 days at +4° C. (T21d4° C.) and in accelerated conditions(T10d4° C.+6d37° C.). In the drawings, FIG. 1 illustrates T0 data.

Adsorption of detoxified pneumolysin to aluminium phosphate underdifferent conditions was studied. The Results are shown in FIG. 1. FIG.1 compares completeness of adsorption of detoxified pneumolysin (dPly)onto aluminium phosphate under different pHs and ratio of dPly:Al³⁺(from aluminium phosphate): (i) pH5.5-6.1 and ratio 1:1, (ii) pH5.5-6.1and ratio 1:2, (iii) pH5.5 to 6.1, ratio 1:3, and (iv) pH6.5 and ratio1:3 at T0 (the day of formulation). Two different antigen lots weretested: E-DPLY-P14 and DPLYADA007.

Conclusion:

Using the process of Example 1 (pH5.5 to 6.1, ratio of dPly:Al³⁺ (fromaluminium phosphate) of 1:3), completeness was improved of ˜20% comparedto other processes (pH5.5-6.1 and ratio of dPly:Al³⁺ (from aluminiumphosphate) of 1:1; pH5.5-6.1 and ratio of dPly:Al³⁺ (from aluminiumphosphate) of 1:2; pH6.5 and ratio of dPly:Al³⁺ (from aluminiumphosphate) of 1:3) without addition of extra alum. A 96-99% completenessof adsorption was observed with the process of Example 1.

After 1 week storage at 37° C., the dPly remained above 95% adsorbed.

In comparison a 78-83% completeness of adsorption was obtained fromadsorption at pH6.5 according to the following method:

Method:

AlPO₄ (Al³⁺) pre-adjusted to pH 6.5 +/− 0.1 + PO₄ (Na/K₂) 2 mM pH 7.15 +dPly    →(ratio 1 μg dPly/3 μg Al³⁺) ↓(stirring Magnetic—Time (min):5-15—Temp (° C.): Room temp) Check or adjust pH 6.5 +/− 0.1 with NaOH0.05M or 0.5M/HCl 0.03M or 0.3M ↓(stirring Magnetic—Time (min):5-15—Temp (° C.): Room temp) Check or adjust pH 6.5 +/− 0.1 with NaOH0.05M or 0.5M HCl 0.03M or 0.3M ↓stirring Magnetic—Time (min):120-150—Temp (° C.): Room temp) ↓ Maturation: Time (min): for at least 7days—Temp (° C.): +2 to +8—Agitation: no ↓ Sampling Remark: the vaccinebulk is maintained under stirring (130 rpm) during all formulationprocess.

TABLE 4 Ingredients Name Component Concentration Other Antigen dPly 1 μgdPly/3 μg Al³⁺ Al³⁺ from AlPO₄ 1 μg dPly/3 μg Al³⁺ NaCl residual PO₄Na/K₂ NaH₂PO₄ ad to K₂HPO₄ 1000 μg/m of dPly pH 6.5 (+/− 0.1)

Lowry Method:

1. To a 200 μl sample add 200 μl 10% SDS.2. Add 1 ml of mixture A and shake. Rest for 10 minutes.3. Add 100 μl of reagent B and shake. Rest for 30 minutes.4. Place in cuvette and take reading at 750 nm

Mixture  A = 50  ml  2%  Na₂CO₃/NaOH  0.1N + 500µl  potassium  tartrate  2% + 500µl  CuSO₄•5H₂O

Reagent B=Folin diluted ×2 in H₂O

Example 3: Antigenicity

The antigenic activity of adsorbed pneumolysin prepared according toExample 1 (M-dPLY-PO₃ and E-DPLY-P01) was determined according to thefollowing method:

Method:

Antigenic activity was determined based on the ratio between proteincontent by ELISA and protein content by Lowry. Elisa was used to measureantigenicity after desorption as described below:

COATING of the wells: Polyclonal Anti-dPly guinea pig sera purified(1807 μg/ml) to 5 μg/ml were diluted 1/400 in PBS and 100 μl added toeach well of a microtitre plate. The plate was then incubated for 2 h at37° C.

Four washes were then carried out using 0.9% NaCl+0.05% Tween™.

Reference Samples:

-   -   M-dPLY-P03 (534 μg dPly/ml) diluted +/−0.7 μg dPLY/ml in PBS        Tween™ 20 0.05% (dilution 1/800).    -   E-DPLY-P01 (944 μg dPly/ml) diluted +/−0.5 μg PS/ml in PBS        Tween™ 20 0.05% (dilution 1/1800)

The above two reference samples were diluted in PBS Tween™ 20 0.05% toreach a concentration of +/−dPly 0.5 μg/ml and 100 μl was added into thefirst and second well. 100 μl of buffer was added in other wells. A 2fold dilution was performed from second well to 11^(th) well. The platewas maintained for 30 minutes at 25° C.+/−2° C. with agitation.

Four washes were then carried out using 0.9% NaCl+0.05% Tween™.

DETECTION: Detection was carried out using polyclonal rabbit seraanti-dPly diluted 1/1000+1% guinea pig serum negative. The plate wasmaintained for 30 minutes at 25° C.+/−2° C. with agitation.

Four washes were then carried out using 0.9% NaCl+0.05% Tween™.

CONJUGATION: Conjugation was carried out by adding anti rabbit Ig,Horseradish Peroxidase Linked F(ab′) 2 fragment (from donkey (Amersham.NA 9340V) diluted 1/1000+1% guinea pig serum negative to the plate(negative control). The plate was maintained for 30 minutes at 25°C.+/−2° C. with agitation.

Four washes were then carried out using 0.9% NaCl+0.05% Tween™

SUBSTRATE: OPD (o-Phenylenediamine (dihydrochloride)) (Sigma P8787) wasused as a chromogenic substrate. A 4 mg tablet was dissolved in 9 mlH₂O, and 1 ml citrate buffer 1M pH 4.2, and 5 μl H₂O₂ were added. 100 μlof the substrate solution was added to each well of the microtitreplate. The plate was maintained for 15 minutes at room temperature inthe absence of light.

The reaction was stopped using 50 μl H₂SO₄ 1N. Spectrophotometer readingwas carried out at 490 nm and 620 nm.

Calculation: Use of 4 parameters method via SoftMaxPro software. Onlythe values between 25 and 85% of reference and samples curves were takeninto consideration (higher and lower asymptote of the curve)

The results are shown in FIG. 2. FIG. 2 shows Elisa recovery for dPlyadsorbed onto aluminium phosphate at pH5.5 to 6.1, ratio of dPly:Al³⁺(from aluminium phosphate) of 1:3. The bars on the left correspond tothe two different antigen lots: dPly E-DPLY-P14 and on the rightcorrespond to dPly DPLYADA007.

Conclusion:

Using the process of Example 1 (pH5.5 to 6.1, ratio of dPly:Al³⁺ (fromaluminium phosphate) of 1:3), the Elisa recovery was within theacceptable level.

Example 4: Particle Size

Method: Particle size was measured by SLS (static light scattering)using a Hydro 2000 μP dispersant unit (Malvern Instruments) at 20-25° C.for 20s using a circulation pump speed of 1500 rpm. Latex polymermicrospheres 5 μm were used as a size standard. Five measurements wereused to calculate the average size distributions for each sample

The results are shown in FIG. 3. FIG. 3 compares the percentage ofparticles of dPly adsorbed onto aluminium phosphate less than 10 μm forunder different pH and ratios of dPly:Al³⁺ (from aluminium phosphate):(i) pH5.5-6.1 and ratio 1:1 and (ii) pH5.5 to 6.1, ratio 1:3. The barsfrom left to right correspond to T0 (time=zero), T7d4° C. (7 days at 4°C.), T7d37° C. (7 days at 37° C.), T10d4+6d37° C. (10 days at 4° C. and6 days at 37° C.) and T21d4° C. (21 days at 4° C.).

Conclusion:

Using the process of Example 1 (pH5.5 to 6.1, ratio of ratio ofdPly:Al³⁺ (from aluminium phosphate) of 1:3), on average >95% ofparticles of detoxified pneumolysin adsorbed onto aluminium phosphatewere <10 μm, within the acceptable level.

Example 5: Adsorption of Conjugated Pneumolysin and PhtD onto AluminiumPhosphate

The preparation of the adsorbed conjugate monobulks consisted of theseparate adsorption of each of the sterile purified conjugate monobulksonto AlPO₄ in a ratio of 1.0 μg conjugate to 10 μg Al³⁺ (as presented inTable 3) according to the method shown below. The purified conjugatemonobulks were mixed to the AlPO₄ (previously adjusted to the serotypespecific adsorption pH), and the sodium chloride 150 mM solution. Themixture was stirred during 15-45 minutes at room temperature (RT).

The mixture was next adjusted at a pH ranging from 5.2 to 6.1 (see Table3) and gently stirred for 2 hours at room temperature for the adsorptionto occur.

A final pH adjustment to 6.1±0.1 took place before storage of theadsorbed conjugate monobulks at 2-8° C. Maturation of the adsorbedconjugate monobulks lasted at least 7 days at 2-8° C.

Method:

AlPO₄ (pH preadjusted as described in Table 5)

+

Diluent NaCl 150 mM

↓

Stirring until homogenization at RT (room temperature)

↓

Add Conjugate bulk

↓

Stirring until homogenization at RT

↓

Adjust pH (pH adjustment serotype specific, Table 5)

↓

Stirring 2 h±15 min at RT

↓

Adjust pH 6.1±0.1 pH

↓

Maturation minimum 7 days at 2-8° C.

↓

Storage at 2-8° C.

TABLE 5 Serotype conjugate pH adjustment PS/Al³⁺ ratio PS19A-dPly 6.11/10 PS22F-PhtD 6.1 1/10Identity S. pneumoniae Polysaccharides by ELISA:

The samples were centrifuged and the supernatants collected and storedat 2-8° C. before use. Analysis was done on the supernatant. For19A-dPly monobulks, the microtiter plates were coated with anti-Plyguinea-pig polyclonal antibodies and incubated for 2 hours at 37° C. andfor 22F-PhtD monobulks, the microtiter plates were coated with anti-PhtDguinea-pig polyclonal antibodies and incubated for 2 hours at 37° C.Plates were washed with NaCl solution containing 0.05% of polysorbate 20(Na Tween20) after each incubation step. After washing of the plate,serial dilutions of the standard material, the internal control of thesupernatant samples was prepared in phosphate-buffered saline solutionsupplemented with 0.05% of polysorbate 20 (PBS Tween 20). These serialdilutions were tested in duplicate. The plates are incubated overnightat 2-8° C. After washing with the NaTween 20 solution, the microtiterplates were incubated. For 19A-dPly incubation was done with rabbitanti-PS polyclonal antibodies, supplemented, if necessary, with negativeguinea-pig serum for 30 minutes at 25° C. For 22F-PhtD incubation wasdone with rabbit anti-PS polyclonal antibodies, supplemented, ifnecessary, with negative guinea-pig serum for 30 minutes at 25° C.Following washing, the captured 19A-dPly antigen was incubated with goatanti-rabbit IgG conjugated to peroxidase, supplemented, if necessary,with negative guinea pig serum for 30 minutes at 25° C. The captured22F-PhtD was incubated with a goat anti-rabbit IgG conjugated toperoxidase, supplemented, if necessary, with negative guinea-pig serumfor 30 minutes at 25° C. After washing, the enzyme substrate,orthophenylenediamine supplemented with H₂O₂ 30%, is added. Afterincubation in the dark for 15 minutes at room temperature, the reactionwas stopped with H₂SO₄ 1.0 N. The absorbance was measured byspectrophotometry at 490 nm and 620 nm. The identity was positive whenthe absorbances were higher than those of the background.

Total Polysaccharide Content by Resorcinol:

Dilutions of the standard material, a lot of purified polysaccharide(PS) of the concerned serotype, in NaCl 150 mM were used to establishthe standard curve. The adsorbed monobulk test samples were diluted inNaCl 150 mM in order to have a content falling within the range of thestandard curve. The resorcinol reagent and the sulfuric acid were addedto each of the samples. After homogenization, the samples were incubatedfor 30 min at 100° C. For the serotype 4 and 5, the samples areincubated for 60 minutes at 100° C. The temperature of the samples wasthen decreased to room temperature for 30 min.

The yellow-orange colour was measured by spectrophotometry at 430 nm.

The PS content was calculated from the PS standard curve.

Completeness of Adsorption to Adjuvant (% Unbound PS):

The completeness of adsorption was determined on adsorbed monobulks byan Enzyme Linked Immunosorbent Assay (ELISA). The assay was ananti-carrier/anti-PS ELISA and the same as the one employed for theidentity testing. After centrifugation of the adsorbed monobulks, theunadsorbed conjugates present in the supernatant were measured by asuitable ELISA (an anti-dPly/anti-PS, anti-PhtD/anti-PS). Thecompleteness of adsorption was expressed in % (amount of the conjugatemeasured in the supernatant to the total of PS content of the adsorbedmonobulks measured by the resorcinol test).

TABLE 6 Tests for the adsorbed PS19A-dPly bulk Tests D19AFAA001 IdentityS. pneumoniae Positive polysaccharide 19A-dPly conjugate by ELISA FreeS. pneumoniae <1.0% polysaccharide type 19A content by ELISACompleteness of adsorption <1.0% to adjuvant (% unbound S. pneumoniaepolysaccharide 19A-dPly conjugate)

TABLE 7 Tests for the adsorbed PS22F-PhtD bulk Tests D22FHAA001 IdentityS. pneumoniae Positive polysaccharide 22F-PhtD conjugate by ELISA FreeS. pneumoniae <1.0 % polysaccharide type 22F content by ELISACompleteness of adsorption <1.0 % to adjuvant (% unbound S. pneumoniaepolysaccharide 19A-dPly conjugate)

GenBank EF413952 SEQ ID NO.: 1MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND GenBank EF413953 SEQ ID NO.: 2MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND GenBank EF413954 SEQ ID NO.: 3MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND GenBank EF413955 SEQ ID NO.: 4MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND GenBank EF413959 (ply-2) SEQ ID NO.: 5MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO 6:MetLysLeuLysThrLeuAlaLeuSerLeuLeuAlaAlaGlyValLeuAlaGlyCysSerSerHisSerSerAsnMetAlaAsnThrGlnMetLysSerAspLysIleIleIleAlaHisArgGlyAlaSerGlyTyrLeuProGluHisThrLeuGluSerLysAlaLeuAlaPheAlaGlnGlnAlaAspTyrLeuGluGlnAspLeuAlaMetThrLysAspGlyArgLeuValValIleHisAspHisPheLeuAspGlyLeuThrAspValAlaLysLysPheProHisArgHisArgLysAspGlyArgTyrTyrValIleAspPheThrLeuLysGluIleGlnSerLeuGluMetThrGluAsnPheGluThrLysAspGlyLysGlnAlaGlnValTyrProAsnArgPheProLeuTrpLysSerHisPheArgIleHisThrPheGluAspGluIleGluPheIleGlnGlyLeuGluLysSerThrGlyLysLysValGlyIleTyrProGluIleLysAlaProTrpPheHisHisGlnAsnGlyLysAspIleAlaAlaGluThrLeuLysValLeuLysLysTyrGlyTyrAspLysLysThrAspMetValTyrLeuGlnThrPheAspPheAsnGluLeuLysArgIleLysThrGluLeuLeuProGlnMetGlyMetAspLeuLysLeuValGlnLeuIleAlaTyrThrAspIrpLysGluThrGlnGluLysAspProLysGlyTyrTrpValAsnTyrAsnTyrAspTrpMetPheLysProGlyAlaMetAlaGluValValLysTyrAlaAspGlyValGlyProGlyTrpTyrMetLeuValAsnLysGluGluSerLysProAspAsnIleValTyrThrProLeuValLysGluLeuAlaGlnTyrAsnValGluValHisProTyrThrValArgLysAspAlaLeuProGluPhePheThrAspValAsnGlnMetTyrAspAlaLeuLeuAsnLysSerGlyAlaThrGlyValPheThrAspPheProAspThrGlyValGluPheLeuLysGlyIleLys SEQ ID NO. 7:MetAspProSerSerHisSerSerAsnMetAlaAsnThrGlnMetLysSerAspLysIleIleIleAlaHisArgGlyAlaSerGlyTyrLeuProGluHisThrLeuGluSerLysAlaLeuAlaPheAlaGlnGlnAlaAspTyrLeuGluGlnAspLeuAlaMetThrLysAspGlyArgLeuValValIleHisAspHisPheLeuAspGlyLeuThrAspValAlaLysLysPheProHisArgHisArgLysAspGlyArgTyrTyrValIleAspPheThrLeuLysGluIleGlnSerLeuGluMetThrGluAsnPheGluThrLysAspGlyLysGlnAlaGlnValTyrProAsnArgPheProLeuTrpLysSerHisPheArgIleHisThrPheGluAspGluIleGluPheIleGlnGlyLeuGluLysSerThrGlyLysLysValGlyIleTyrProGluIleLysAlaProTrpPheHisHisGlnAsnGlyLysAspIleAlaAlaGluThrLeuLysValLeuLysLysTyrGlyTyrAspLysLysThrAspMetValTyrLeuGlnThrPheAspPheAsnGluLeuLysArgIleLysThrGluLeuLeuProGlnMetGlyMetAspLeuLysLeuValGlnLeuIleAlaTyrThrAspIrpLysGluThrGlnGluLysAspProLysGlyTyrTrpValAsnTyrAsnTyrAspTrpMetPheLysProGlyAlaMetAlaGluValValLysTyrAlaAspGlyValGlyProGlyTrpTyrMetLeuValAsnLysGluGluSerLysProAspAsnIleValTyrThrProLeuValLysGluLeuAlaGlnTyrAsnValGluValHisProTyrThrValArgLysAspAlaLeuProGluPhePheThrAspValAsnGlnMetTyrAspAlaLeuLeuAsnLysSerGlyAlaThrGlyValPheThrAspPheProAspThrGlyValGluPheLeuLysGlyIleLys SEQ ID NO. 8:SerSerHisSerSerAsnMetAlaAsnThr

1. An immunogenic composition comprising detoxified pneumolysin adsorbedonto aluminium phosphate, wherein more than 85% of the detoxifiedpneumolysin is adsorbed onto aluminium phosphate.
 2. An immunogeniccomposition according to claim 1, wherein more than 95% of thedetoxified pneumolysin is adsorbed onto aluminium phosphate.
 3. Animmunogenic composition according to claim 1 or claim 2, wherein theimmunogenic composition has a pH between 6 and
 7. 4. An immunogeniccomposition according to claim 1, wherein greater than 80% of thedetoxified pneumolysin adsorbed onto aluminium phosphate has a particlesize less than 10 μm.
 5. An immunogenic composition according to claim1, wherein greater than 85% of the detoxified pneumolysin adsorbed ontoaluminium phosphate has a particle size less than 10 μm.
 6. Animmunogenic composition according to claim 1, wherein the detoxifiedpneumolysin has been chemically detoxified.
 7. An immunogeniccomposition according to claim 1, wherein the detoxified pneumolysin hasbeen genetically detoxified.
 8. An immunogenic composition according toclaim 3, wherein the detoxified pneumolysin is unconjugated orconjugated to a saccharide.
 9. An immunogenic composition according toclaim 8, wherein the detoxified pneumolysin is conjugated to asaccharide, wherein the saccharide is a capsular saccharide of S.pneumoniae.
 10. An immunogenic composition according to claim 1, furthercomprising PhtD adsorbed onto aluminium phosphate.
 11. An immunogeniccomposition according to claim 10, wherein the PhtD is unconjugated. 12.An immunogenic composition according to claim 10, wherein the PhtD isconjugated to a saccharide, for example a capsular saccharide of S.pneumoniae.
 13. An immunogenic composition according to claim 1 or claim2, further comprising 10 or more S. pneumoniae capsular polysaccharidesfrom different S. pneumoniae serotypes conjugated to a carrier protein.14. (canceled)
 15. (canceled)
 16. (canceled)
 17. (canceled) 18.(canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. Animmunogenic composition according to claim 1, prepared by a process foradsorption of detoxified pneumolysin onto aluminium phosphate comprising(i) admixing detoxified pneumolysin and the aluminium phosphate at a pHless than 6.5 and (ii) mixing the adsorbed detoxified pneumolysin withone or more antigens(s) other than detoxified pneumolysin.
 28. A vaccinecomprising the immunogenic composition of claim 1 or claim 27 and apharmaceutically acceptable excipient or carrier.
 29. A method for thetreatment or prevention of Streptococcus pneumoniae infection in asubject in need thereof (e.g. human) comprising administering to saidsubject a therapeutically effective amount of an immunogenic compositionof claim
 1. 30. A method of immunising a human host againstStreptococcus pneumoniae infection comprising administering to the hostan immunoprotective dose of the immunogenic composition of claim
 1. 31.A method of inducing an immune response to Streptococcus pneumoniae in asubject (e.g. human), the method comprising administering atherapeutically or prophylactically effective amount of the immunogeniccomposition of claim
 1. 32. (canceled)
 33. (canceled)